Cold-adapted equine influenza viruses

ABSTRACT

The present invention provides experimentally-generated cold-adapted equine influenza viruses, and reassortant influenza A viruses comprising at least one genome segment of such an equine influenza virus, wherein the equine influenza virus genome segment confers at least one identifying phenotype of the cold-adapted equine influenza virus, such as cold-adaptation, temperature sensitivity, dominant interference, or attenuation. Such viruses are formulated into therapeutic compositions to protect animals from diseases caused by influenza A viruses, and in particular, to protect horses from disease caused by equine influenza virus. The present invention also includes methods to protect animals from diseases caused by influenza A virus utilizing the claimed therapeutic compositions. Such methods include using a therapeutic composition as a vaccine to generate a protective immune response in an animal prior to exposure to a virulent virus, and using a therapeutic composition as a treatment for an animal that has been recently infected with a virulent virus, or is likely to be subsequently exposed to virulent virus in a few days whereby the therapeutic composition interferes with the growth of the virulent virus, even in the absence of immunity. The present invention also provides methods to produce cold-adapted equine influenza viruses, and reassortant influenza A viruses having at least one genome segment of an equine influenza virus generated by cold-adaptation.

CROSS-REFERENCE TO RELATED APPLICATION

This application C-I-P U.S. patent application Ser. No. 09/133,921,filed Aug. 13, 1998, now U.S. Pat. No. 6,177,082 which is a C-I-P ofPCT/US99/18583, filed Aug. 12, 1999; each entitled COLD-ADAPTED EQUINEINFLUENZA VIRUSES. The patent applications referred to in this sectionare incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The present invention relates to experimentally-generated cold-adaptedequine influenza viruses, and particularly to cold-adapted equineinfluenza viruses having additional phenotypes, such as attenuation,dominant interference, or temperature sensitivity. The invention alsoincludes reassortant influenza A viruses which contain at least onegenome segment from such an equine influenza virus, such that thereassortant virus includes certain phenotypes of the donor equineinfluenza virus. The invention further includes genetically-engineeredequine influenza viruses, produced through reverse genetics, whichcomprise certain identifying phenotypes of a cold-adapted equineinfluenza virus of the present invention. The present invention alsorelates to the use of these viruses in therapeutic compositions toprotect animals from diseases caused by influenza viruses.

BACKGROUND OF THE INVENTION

Equine influenza virus has been recognized as a major respiratorypathogen in horses since about 1956. Disease symptoms caused by equineinfluenza virus can be severe, and are often followed by secondarybacterial infections. Two subtypes of equine influenza virus arerecognized, namely subtype-1, the prototype being A/Equine/Prague/1/56(H7N7), and subtype-2, the prototype being A/Equine/Miami/1/63 (H3N8).Presently, the predominant virus subtype is subtype-2, which has furtherdiverged among Eurasian and North American isolates in recent years.

The currently licensed vaccine for equine influenza is an inactivated(killed) virus vaccine. This vaccine provides minimal, if any,protection for horses, and can produce undesirable side effects, forexample, inflammatory reactions at the site of injection. See, e.g.,Mumford, 1987, Equine Infectious Disease IV, 207-217, and Mumford, etal., 1993, Vaccine 11, 1172-1174. Furthermore, current modalities cannotbe used in young foals, because they cannot overcome maternal immunity,and can induce tolerance in a younger animal. Based on the severity ofdisease, there remains a need for safe, effective therapeuticcompositions to protect horses against equine influenza disease.

Production of therapeutic compositions comprising cold-adapted humaninfluenza viruses is described, for example, in Maassab, et al., 1960,Nature 7,612-614, and Maassab, et al., 1969, J. Immunol. 102, 728-732.Furthermore, these researchers noted that cold-adapted human influenzaviruses, i.e., viruses that have been adapted to grow at lower thannormal temperatures, tend to have a phenotype wherein the virus istemperature sensitive; that is, the virus does not grow well at certainhigher, non-permissive temperatures at which the wild-type virus willgrow and replicate. Various cold-adapted human influenza A viruses,produced by reassortment with existing cold-adapted human influenza Aviruses, have been shown to elicit good immune responses in vaccinatedindividuals, and certain live attenuated cold-adapted reassortant humaninfluenza A viruses have proven to protect humans against challenge withwild-type virus. See, e.g., Clements, et al., 1986, J. Clin. Microbiol.23, 73-76. In U.S. Pat. No. 5,149,531, by Youngner, et al., issued Sep.22, 1992, the inventors of the present invention further demonstratedthat certain reassortant cold-adapted human influenza A viruses alsopossess a dominant interference phenotype, i.e., they inhibit the growthof their corresponding parental wild-type strain, as well asheterologous influenza A viruses.

U.S. Pat. No. 4,683,137, by Coggins et al., issued Jul. 28, 1987, andU.S. Pat. No. 4,693,893, by Campbell, issued Sep. 15, 1987, discloseattenuated therapeutic compositions produced by reassortment ofwild-type equine influenza viruses with attenuated, cold-adapted humaninfluenza A viruses. Although these therapeutic compositions appear tobe generally safe and effective in horses, they pose a significantdanger of introducing into the environment a virus containing both humanand equine influenza genes.

SUMMARY OF THE INVENTION

The present invention provides experimentally-generated cold-adaptedequine influenza viruses, reassortant influenza A viruses that compriseat least one genome segment of an equine influenza virus generated bycold-adaptation such that the equine influenza virus genome segmentconfers at least one identifying phenotype of a cold-adapted equineinfluenza virus on the reassortant virus, and genetically-engineeredequine influenza viruses, produced through reverse genetics, whichcomprise at least one identifying phenotype of a cold-adapted equineinfluenza virus. Identifying phenotypes include cold-adaptation,temperature sensitivity, dominant interference, and attenuation. Theinvention further provides a therapeutic composition to protect ananimal against disease caused by an influenza A virus, where thetherapeutic composition includes a cold-adapted equine influenza virus areassortant influenza A virus, or a genetically-engineered equineinfluenza virus of the present invention. Also provided is a method toprotect an animal from diseases caused by an influenza A virus whichincludes the administration of such a therapeutic composition. Alsoprovided are methods to produce a cold-adapted equine influenza virus,and methods to produce a reassortant influenza A virus which comprisesat least one genome segment of a cold-adapted equine influenza virus,where the equine influenza genome segment confers on the reassortantvirus at least one identifying phenotype of the cold-adapted equineinfluenza virus.

A cold-adapted equine influenza virus is one that replicates inembryonated chicken eggs at a temperature ranging from about 26° C. toabout 30° C. Preferably, a cold-adapted equine influenza virus,reassortant influenza A virus, or genetically-engineered equineinfluenza virus of the present invention is attenuated, such that itwill not cause disease in a healthy animal.

In one embodiment, a cold-adapted equine influenza virus, reassortantinfluenza A virus, or genetically-engineered equine influenza virus ofthe present invention is also temperature sensitive, such that the virusreplicates in embryonated chicken eggs at a temperature ranging fromabout 26° C. to about 30° C., forms plaques in tissue culture cells at apermissive temperature of about 34° C., but does not form plaques intissue culture cells at a non-permissive temperature of about 39° C.

In one embodiment, such a temperature sensitive virus comprises twomutations: a first mutation that inhibits plaque formation at atemperature of about 39° C., that mutation co-segregating with thegenome segment that encodes the viral nucleoprotein gene; and a secondmutation that inhibits all viral protein synthesis at a temperature ofabout 39° C.

In another embodiment, a cold-adapted, temperature sensitive equineinfluenza virus of the present invention replicates in embryonatedchicken eggs at a temperature ranging from about 26° C. to about 30° C.,forms plaques in tissue culture cells at a permissive temperature ofabout 34° C., but does not form plaques in tissue culture cells orexpress late viral proteins at a non-permissive temperature of about 37°C.

Typically, a cold-adapted equine influenza virus of the presentinvention is produced by passaging a wild-type equine influenza virusone or more times, and then selecting viruses that stably grow andreplicate at a reduced temperature. A cold-adapted equine influenzavirus produced thereby includes, in certain embodiments, a dominantinterference phenotype, that is, the virus, when co-infected with aparental equine influenza virus or heterologous wild-type influenza Avirus, will inhibit the growth of that virus.

Examples of cold-adapted equine influenza viruses of the presentinvention include EIV-P821, identified by accession No. ATCC VR-2625;EIV-P824, identified by accession No. ATCC VR-2624; EIV-MSV+5,identified by accession No. ATCC VR-627; and progeny of such viruses.

Therapeutic compositions of the present invention include from about 10⁵TCID₅₀ units to about 10⁸ TCID₅₀ units, and preferably about 2×10⁶TCID₅₀ units, of a cold-adapted equine influenza virus, reassortantinfluenza A virus, or genetically-engineered equine influenza virus ofthe present invention.

The present invention also includes a method to protect an animal fromdisease caused by an influenza A virus, which includes the step ofadministering to the animal a therapeutic composition including acold-adapted equine influenza virus, a reassortant influenza A virus, ora genetically-engineered equine influenza virus of the presentinvention. Preferred animals to protect include equids, with horses andponies being particularly preferred.

Yet another embodiment of the present invention is a method to generatea cold-adapted equine influenza virus. The method includes the steps ofpassaging a wild-type equine influenza virus; and selecting viruses thatgrow at a reduced temperature. In one embodiment, the method includesrepeating the passaging and selection steps one or more times, whileprogressively reducing the temperature. Passaging of equine influenzavirus preferably takes place in embryonated chicken eggs.

Another embodiment is an method to produce a reassortant influenza Avirus through genetic reassortment of the genome segments of a donorcold-adapted equine influenza virus of the present invention with thegenome segments of a recipient influenza A virus. Reassortant influenzaA viruses of the present invention are produced by a method thatincludes the steps of: (a) mixing the genome segments of a donorcold-adapted equine influenza virus with the genome segments of arecipient influenza A virus, and (b) selecting viruses which include atleast one identifying phenotype of the donor equine influenza virus.Identifying phenotypes include cold-adaptation, temperature sensitivity,dominant interference, and attenuation. Preferably, such reassortantviruses at least include the attenuation phenotype of the donor virus. Atypical reassortant virus will have the antigenicity of the recipientvirus, that is, it will retain the hemagglutinin (HA) and neuraminidase(NA) phenotypes of the recipient virus.

The present invention further provides methods to propagate cold-adaptedequine influenza viruses or reassortant influenza A viruses of thepresent invention. These methods include propagation in embryonatedchicken eggs or in tissue culture cells.

The present invention also describes nucleic acid molecules encodingwild-type and cold-adapted equine influenza proteins M, HA, NS, PB2,PB2-N, PB2-C, PB1, PB1-N, PB1-C, and PA-C. One embodiment of the presentinvention is an isolated equine nucleic acid molecule having a nucleicacid sequence selected from a group consisting of SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18,SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25 SEQID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ IDNO:59, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:67, SEQ IDNO:68, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:76, SEQ ID NO:78, SEQ IDNO:79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:85, SEQ ID NO:87, SEQ IDNO:88, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ IDNO:96, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:106 and SEQ ID NO:108 anda nucleic acid molecule comprising a nucleic acid sequence which isfully complementary to any of such nucleic acid sequences. Anotherembodiment of the present invention is an isolated equine nucleic acidmolecule that encodes a protein comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ IDNO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ IDNO:24, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:55, SEQ IDNO:58, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:77, SEQ IDNO:81, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, SEQ IDNO:104 and SEQ ID NO:107. Another embodiment is an isolated equineinfluenza protein that comprises an amino acid sequence selected from agroup consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11,SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:45,SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:63,SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:81, SEQ ID NO:86,SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:104 and SEQ IDNO:107. Also included in the present invention is a virus that includeany of these nucleic acid molecules or proteins. In one embodiment, sucha virus is equine influenza virus or a reassortant virus.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides experimentally-generated cold-adaptedequine influenza viruses comprising certain defined phenotypes, whichare disclosed herein. It is to be noted that the term “a” or “an”entity, refers to one or more of that entity; for example, “acold-adapted equine influenza virus” can include one or morecold-adapted equine influenza viruses. As such, the terms “a” (or “an”),“one or more,” and “at least one” can be used interchangeably herein. Itis also to be noted that the terms “comprising,” “including,” and“having” can be used interchangeably. Furthermore, an item “selectedfrom the group consisting of” refers to one or more of the items in thatgroup, including combinations thereof.

A cold-adapted equine influenza virus of the present invention is avirus that has been generated in the laboratory, and as such, is not avirus as occurs in nature. Since the present invention also includesthose viruses having the identifying phenotypes of such a cold-adaptedequine influenza virus, an equine influenza virus isolated from amixture of naturally-occurring viruses, i.e., removed from its naturalmilieu, but having the claimed phenotypes, is included in the presentinvention. A cold-adapted equine influenza virus of the presentinvention does not require any specific level of purity. For example, acold-adapted equine influenza virus grown in embryonated chicken eggsmay be in a mixture with the allantoic fluid (AF), and a cold-adaptedequine influenza virus grown in tissue culture cells may be in a mixturewith disrupted cells and tissue culture medium.

As used herein, an “equine influenza virus” is an influenza virus thatinfects and grows in equids, e.g., horses or ponies. As used herein,“growth” of a virus denotes the ability of the virus to reproduce or“replicate” itself in a permissive host cell. As such, the terms,“growth of a virus” and “replication of a virus” are usedinterchangeably herein. Growth or replication of a virus in a particularhost cell can be demonstrated and measured by standard methodswell-known to those skilled in the art of virology. For example, samplescontaining infectious virus, e.g., as contained in nasopharyngealsecretions from an infected horse, are tested for their ability to causecytopathic effect (CPE), e.g., virus plaques, in tissue culture cells.Infectious virus may also be detected by inoculation of a sample intothe allantoic cavity of embryonated chicken eggs, and then testing theAF of eggs thus inoculated for its ability to agglutinate red bloodcells, i.e., cause hemagglutination, due to the presence of theinfluenza virus hemagglutinin (HA) protein in the AF.

Naturally-occurring, i.e., wild-type, equine influenza viruses replicatewell at a temperature from about 34° C. to about 39° C. For example,wild-type equine influenza virus replicates in embryonated chicken eggsat a temperature of about 34° C., and replicates in tissue culture cellsat a temperature from about 34° C. to about 39° C. As used herein, a“cold-adapted” equine influenza virus is an equine influenza virus thathas been adapted to grow at a temperature lower than the optimal growthtemperature for equine influenza virus. One example of a cold-adaptedequine influenza virus of the present invention is a virus thatreplicates in embryonated chicken eggs at a temperature of about 30° C.A preferred cold-adapted equine influenza virus of the present inventionreplicates in embryonated chicken eggs at a temperature of about 28° C.Another preferred cold-adapted equine influenza virus of the presentinvention replicates in embryonated chicken eggs at a temperature ofabout 26° C. In general, preferred cold-adapted equine influenza virusesof the present invention replicate in embryonated chicken eggs at atemperature ranging from about 26° C. to about 30° C., i.e., at a rangeof temperatures at which a wild-type virus will grow poorly or not atall. It should be noted that the ability of such viruses to replicatewithin that temperature range does not preclude their ability to alsoreplicate at higher or lower temperatures. For example, one embodimentis a cold-adapted equine influenza virus that replicates in embryonatedchicken eggs at a temperature of about 26° C., but also replicates intissue culture cells at a temperature of about 34° C. As with wild-typeequine influenza viruses, cold-adapted equine influenza viruses of thepresent invention generally form plaques in tissue culture cells, forexample Madin Darby Canine Kidney Cells (MDCK) at a temperature of about34° C. Examples of suitable and preferred cold-adapted equine influenzaviruses of the present invention are disclosed herein.

One embodiment of the present invention is a cold-adapted equineinfluenza virus that is produced by a method which includes passaging awild-type equine influenza virus, and then selecting viruses that growat a reduced temperature. Cold-adapted equine influenza viruses of thepresent invention can be produced, for example, by sequentiallypassaging a wild-type equine influenza virus in embryonated chicken eggsat progressively lower temperatures, thereby selecting for certainmembers of the virus mixture which stably replicate at the reducedtemperature. An example of a passaging procedure is disclosed in detailin the Examples section. During the passaging procedure, one or moremutations appear in certain of the single-stranded RNA segmentscomprising the influenza virus genome, which alter the genotype, i.e.,the primary nucleotide sequence of those RNA segments. As used herein, a“mutation” is an alteration of the primary nucleotide sequence of anygiven RNA segment making up an influenza virus genome. Examples ofmutations include substitution of one or more nucleotides, deletion ofone or more nucleotides, insertion of one or more nucleotides, orinversion of a stretch of two or more nucleotides. By selecting forthose members of the virus mixture that stably replicate at a reducedtemperature, a virus with a cold-adaptation phenotype is selected. Asused herein, a “phenotype” is an observable or measurable characteristicof a biological entity such as a cell or a virus, where the observedcharacteristic is attributable to a specific genetic configuration ofthat biological entity, i.e., a certain genotype. As such, acold-adaptation phenotype is the result of one or more mutations in thevirus genome. As used herein, the terms “a mutation,” “a genome,” “agenotype,” or “a phenotype” refer to one or more, or at least onemutation, genome, genotype, or phenotype, respectively.

Additional, observable phenotypes in a cold-adapted equine influenzavirus may occur, and will generally be the result of one or moreadditional mutations in the genome of such a virus. For example, acold-adapted equine influenza virus of the present invention may, inaddition, be attenuated, exhibit dominant interference, and/or betemperature sensitive.

In one embodiment, a cold-adapted equine influenza virus of the presentinvention has a phenotype characterized by attenuation. A cold-adaptedequine influenza virus is “attenuated,” when administration of the virusto an equine influenza virus-susceptible animal results in reduced orabsent clinical signs in that animal, compared to clinical signsobserved in animals that are infected with wild-type equine influenzavirus. For example, an animal infected with wild-type equine influenzavirus will display fever, sneezing, coughing, depression, and nasaldischarges. In contrast, an animal administered an attenuated,cold-adapted equine influenza virus of the present invention willdisplay minimal or no, i.e., undetectable, clinical disease signs.

In another embodiment, a cold-adapted equine influenza virus of thepresent invention comprises a temperature sensitive phenotype. As usedherein, a temperature sensitive cold-adapted equine influenza virusreplicates at reduced temperatures, but no longer replicates or formsplaques in tissue culture cells at certain higher growth temperatures atwhich the wild-type virus will replicate and form plaques. While notbeing bound by theory, it is believed that replication of equineinfluenza viruses with a temperature sensitive phenotype is largelyrestricted to the cool passages of the upper respiratory tract, and doesnot replicate efficiently in the lower respiratory tract, where thevirus is more prone to cause disease symptoms. A temperature at which atemperature sensitive virus will grow is referred to herein as a“permissive” temperature for that temperature sensitive virus, and ahigher temperature at which the temperature sensitive virus will notgrow, but at which a corresponding wild-type virus will grow, isreferred to herein as a “non-permissive” temperature for thattemperature sensitive virus. For example, certain temperature sensitivecold-adapted equine influenza viruses of the present invention replicatein embryonated chicken eggs at a temperature at or below about 30° C.,preferably at about 28° C. or about 26° C., and will form plaques intissue culture cells at a permissive temperature of about 34° C., butwill not form plaques in tissue culture cells at a non-permissivetemperature of about 39° C. Other temperature sensitive cold-adaptedequine influenza viruses of the present invention replicate inembryonated chicken eggs at a temperature at or below about 30° C.,preferably at about 28° C. or about 26° C., and will form plaques intissue culture cells at a permissive temperature of about 34° C., butwill not form plaques in tissue culture cells at a non-permissivetemperature of about 37° C.

Certain cold-adapted equine influenza viruses of the present inventionhave a dominant interference phenotype; that is, they dominate aninfection when co-infected into cells with another influenza A virus,thereby impairing the growth of that other virus. For example, when acold-adapted equine influenza virus of the present invention, having adominant interference phenotype, is co-infected into MDCK cells with thewild-type parental equine influenza virus, A/equine/Kentucky/1/91(H3N8), growth of the parental virus is impaired. Thus, in an animalthat has recently been exposed to, or may be soon exposed to, a virulentinfluenza virus, i.e., an influenza virus that causes disease symptoms,administration of a therapeutic composition comprising a cold-adaptedequine influenza virus having a dominant interference phenotype into theupper respiratory tract of that animal will impair the growth of thevirulent virus, thereby ameliorating or reducing disease in that animal,even in the absence of an immune response to the virulent virus.

Dominant interference of a cold-adapted equine influenza virus having atemperature sensitive phenotype can be measured by standard virologicalmethods. For example, separate monolayers of MDCK cells can be infectedwith (a) a virulent wild-type influenza A virus, (b) a temperaturesensitive, cold-adapted equine influenza virus, and (c) both viruses ina co-infection, with all infections done at multiplicities of infection(MOI) of about 2 plaque forming units (pfu) per cell. After infection,the virus yields from the various infected cells are measured byduplicate plaque assays performed at the permissive temperature for thecold-adapted equine influenza virus and at the non-permissivetemperature of that virus. A cold adapted equine influenza virus havinga temperature sensitive phenotype is unable to form plaques at itsnon-permissive temperature, while the wild-type virus is able to formplaques at both the permissive and non-permissive temperatures. Thus itis possible to measure the growth of the wild-type virus in the presenceof the cold adapted virus by comparing the virus yield at thenon-permissive temperature of the cells singly infected with wild-typevirus to the yield at the non-permissive temperature of the wild-typevirus in doubly infected cells.

Cold-adapted equine influenza viruses of the present invention arecharacterized primarily by one or more of the following identifyingphenotypes: cold-adaptation, temperature sensitivity, dominantinterference, and/or attenuation. As used herein, the phrase “an equineinfluenza virus comprises the identifying phenotype(s) ofcold-adaptation, temperature sensitivity, dominant interference, and/orattenuation” refers to a virus having such a phenotype(s). Examples ofsuch viruses include, but are not limited to, EIV-P821, identified byaccession No. ATCC VR-2625, EIV-P824, identified by accession No. ATCCVR-2624, and EIV-MSV+5, identified by accession No. ATCC VR-2627, aswell as EIV-MSV0, EIV, MSV+1, EIV-MSV+2, EIV-MSV+3, and EIV-MSV+4.Production of such viruses is described in the examples. For example,cold-adapted equine influenza virus EIV-P821 is characterized by, i.e.,has the identifying phenotypes of, (a) cold-adaptation, e.g., itsability to replicate in embryonated chicken eggs at a temperature ofabout 26° C.; (b) temperature sensitivity, e.g., its inability to formplaques in tissue culture cells and to express late gene products at anon-permissive temperature of about 37° C., and its inability to formplaques in tissue culture cells and to synthesize any viral proteins ata non-permissive temperature of about 39° C.; (c) its attenuation uponadministration to an equine influenza virus-susceptible animal; and (d)dominant interference, e.g., its ability, when co-infected into a cellwith a wild-type influenza A virus, to interfere with the growth of thatwild-type virus. Similarly, cold-adapted equine influenza virus EIV-P824is characterized by (a) cold adaptation, e.g., its ability to replicatein embryonated chicken eggs at a temperature of about 28° C.; (b)temperature sensitivity, e.g., its inability to form plaques in tissueculture cells at a non-permissive temperature of about 39° C.; and (c)dominant interference, e.g., its ability, when co-infected into a cellwith a wild-type influenza A virus, to interfere with the growth of thatwild-type virus. In another example, cold-adapted equine influenza virusEIV-MSV+5 is characterized by (a) cold-adaptation, e.g., its ability toreplicate in embryonated chicken eggs at a temperature of about 26° C.;(b) temperature sensitivity, e.g., its inability to form plaques intissue culture cells at a non-permissive temperature of about 39° C.;and (c) its attenuation upon administration to an equine influenzavirus-susceptible animal.

In certain cases, the RNA segment upon which one or more mutationsassociated with a certain phenotype occur may be determined throughreassortment analysis by standard methods, as disclosed herein. In oneembodiment, a cold-adapted equine influenza virus of the presentinvention comprises a temperature sensitive phenotype that correlateswith at least two mutations in the genome of that virus. In thisembodiment, one of the two mutations, localized by reassortment analysisas disclosed herein, inhibits, i.e., blocks or prevents, the ability ofthe virus to form plaques in tissue culture cells at a non-permissivetemperature of about 39° C. This mutation co-segregates with the segmentof the equine influenza virus genome that encodes the nucleoprotein (NP)gene of the virus, i.e., the mutation is located on the same RNA segmentas the NP gene. In this embodiment, the second mutation inhibits allprotein synthesis at a non-permissive temperature of about 39° C. Assuch, at the non-permissive temperature, the virus genome is incapableof expressing any viral proteins. Examples of cold-adapted equineinfluenza viruses possessing these characteristics are EIV-P821 and EIVMSV+5. EIV-P821 was generated by serial passaging of a wild-type equineinfluenza virus in embryonated chicken eggs by methods described inExample 1A. EIV-MSV+5 was derived by further serial passaging ofEIV-P821, as described in Example 1E.

Furthermore, a cold-adapted, temperature sensitive equine influenzavirus comprising the two mutations which inhibit plaque formation andviral protein synthesis at a non-permissive temperature of about 39° C.can comprise one or more additional mutations, which inhibit the virus'ability to synthesize late gene products and to form plaques in tissueculture cells at a non-permissive temperature of about 37° C. An exampleof a cold-adapted equine influenza virus possessing thesecharacteristics is EIV-P821. This virus isolate replicates inembryonated chicken eggs at a temperature of about 26° C., and does notform plaques or express any viral proteins at a temperature of about 39°C. Furthermore, EIV-P821 does not form plaques on MDCK cells at anon-permissive temperature of about 37° C., and at this temperature,late gene expression is inhibited in such a way that late proteins arenot produced, i.e., normal levels of NP protein are synthesized, reducedor undetectable levels of M1 or HA proteins are synthesized, andenhanced levels of the polymerase proteins are synthesized. Since thisphenotype is typified by differential viral protein synthesis, it isdistinct from the protein synthesis phenotype seen at a non-permissivetemperature of about 39° C., which is typified by the inhibition ofsynthesis of all viral proteins.

Pursuant to 37 CFR §1.802 (a-c), cold-adapted equine influenza viruses,designated herein as EIV-P821, an EIV-P824 were deposited with theAmerican Type Culture Collection (ATCC, 10801 University Boulevard,Manassas, Va. 20110-2209) under the Budapest Treaty as ATCC AccessionNos. ATCC VR-2625, and ATCC VR-2624, respectively, on Jul. 11, 1998.Cold-adapted equine influenza virus EIV-MSV+5 was deposited with theATCC as ATCC Accession No. ATCC VR-2627 on Aug. 3, 1998. Pursuant to 37CFR §1.806, the deposits are made for a term of at least thirty (30)years and at least five (5) years after the most recent request for thefurnishing of a sample of the deposit was received by the depository.Pursuant to 37 CFR §1.808 (a)(2), all restrictions imposed by thedepositor on the availability to the public will be irrevocably removedupon the granting of the patent.

Preferred cold-adapted equine influenza viruses of the present inventionhave the identifying phenotypes of EIV-P821, EIV-P824, and EIV-MSV+5.Particularly preferred cold-adapted equine influenza viruses includeEIV-P821, EIV-P824, EIV-MSV+5, and progeny of these viruses. As usedherein, “progeny” are “offspring,” and as such can slightly alteredphenotypes compared to the parent virus, but retain identifyingphenotypes of the parent virus, for example, cold-adaptation,temperature sensitivity, dominant interference, or attenuation. Forexample, cold-adapted equine influenza virus EIV-MSV+5 is a “progeny” ofcold-adapted equine influenza virus EIV-P821. “Progeny” also includereassortant influenza A viruses that comprise one or more identifyingphenotypes of the donor parent virus.

Reassortant influenza A viruses of the present invention are produced bygenetic reassortment of the genome segments of a donor cold-adaptedequine influenza virus of the present invention with the genome segmentsof a recipient influenza A virus, and then selecting a reassortant virusthat derives at least one of its eight RNA genome segments from thedonor virus, such that the reassortant virus acquires at least oneidentifying phenotype of the donor cold-adapted equine influenza virus.Identifying phenotypes include cold-adaptation, temperature sensitivity,attenuation, and dominant interference. Preferably, reassortantinfluenza A viruses of the present invention derive at least theattenuation phenotype of the donor virus. Methods to isolate reassortantinfluenza viruses are well known to those skilled in the art of virologyand are disclosed, for example, in Fields, et al., 1996, FieldsVirology, 3d ed., Lippincott-Raven; and Palese, et al., 1976, J. Virol.,17, 876-884. Fields, et al., ibid. and Palese, et al., ibid.

A suitable donor equine influenza virus is a cold-adapted equineinfluenza virus of the present invention, for example, EIV-P821,identified by accession No. ATCC VR-2625, EIV-P824, identified byaccession No. ATCC VR-2624, or EIV-MSV+5, identified by accession No.ATCC VR-2627. A suitable recipient influenza A virus can be anotherequine influenza virus, for example a Eurasian subtype 2 equineinfluenza virus such as A/equine/Suffolk/89 (H3N8) or a subtype 1 equineinfluenza virus such as A/Prague/1/56 (H7N7). A recipient influenza Avirus can also be any influenza A virus capable of forming a reassortantvirus with a donor cold-adapted equine influenza virus. Examples of suchinfluenza A viruses include, but are not limited to, human influenzaviruses such as A/Puerto Rico/8/34 (H1N1), A/Hong Kong/156/97 (H5N1),A/Singapore/1/57 (H2N2), and A/Hong Kong/1/68 (H3N2); swine viruses suchas A/Swine/Iowa/15/30 (H1N1); and avian viruses such as A/mallard/NewYork/6750/78 (H2N2) and A/chicken/Hong Kong/258/97 (H5N 1). Areassortant virus of the present invention can include any combinationof donor and recipient gene segments, as long as the resultingreassortant virus possesses at least one identifying phenotype of thedonor virus.

One example of a reassortant virus of the present invention is a “6+2”reassortant virus, in which the six “internal gene segments,” i.e.,those comprising the NP, PB2, PB1, PA, M, and NS genes, are derived fromthe donor cold-adapted equine influenza virus genome, and the two“external gene segments,” i.e., those comprising the HA and NA genes,are derived from the recipient influenza A virus. A resultant virus thusproduced has the attenuated, cold-adapted, temperature sensitive, and/ordominant interference phenotypes of the donor cold-adapted equineinfluenza virus, but the antigenicity of the recipient strain.

In yet another embodiment, a cold-adapted equine influenza virus of thepresent invention can be produced through recombinant means. In thisapproach, one or more specific mutations, associated with identifiedcold-adaptation, attenuation, temperature sensitivity, or dominantinterference phenotypes, are identified and are introduced back into awild-type equine influenza virus strain using a reverse geneticsapproach. Reverse genetics entails using RNA polymerase complexesisolated from influenza virus-infected cells to transcribe artificialinfluenza virus genome segments containing the mutation(s),incorporating the synthesized RNA segment(s) into virus particles usinga helper virus, and then selecting for viruses containing the desiredchanges. Reverse genetics methods for influenza viruses are described,for example, in Enami, et al., 1990, Proc. Natl. Acad. Sci. 87,3802-3805; and in U.S. Pat. No. 5,578,473, by Palese, et al., issuedNov. 26, 1996. This approach allows one skilled in the art to produceadditional cold-adapted equine influenza viruses of the presentinvention without the need to go through the lengthy cold-adaptationprocess, and the process of selecting mutants both in vitro and in vivowith the desired virus phenotype.

A cold-adapted equine influenza virus of the present invention may bepropagated by standard virological methods well-known to those skilledin the art, examples of which are disclosed herein. For example, acold-adapted equine influenza virus can be grown in embryonated chickeneggs or in eukaryotic tissue culture cells. Suitable continuouseukaryotic cell lines upon which to grow a cold-adapted equine influenzavirus of the present invention include those that support growth ofinfluenza viruses, for example, MDCK cells. Other suitable cells uponwhich to grow a cold-adapted equine influenza virus of the presentinvention include, but are not limited to, primary kidney cell culturesof monkey, calf, hamster or chicken.

In one embodiment, the present invention provides a therapeuticcomposition to protect an animal against disease caused by an influenzaA virus, where the therapeutic composition includes either acold-adapted equine influenza virus or a reassortant influenza A viruscomprising at least one genome segment of an equine influenza virusgenerated by cold-adaptation, wherein the equine influenza virus genomesegment confers at least one identifying phenotype of the cold-adaptedequine influenza virus. In addition, a therapeutic composition of thepresent invention can include an equine influenza virus that has beengenetically engineered to comprise one or more mutations, where thosemutations have been identified to confer a certain identifying phenotypeon a cold-adapted equine influenza virus of the present invention. Asused herein, the phrase “disease caused by an influenza A virus” refersto the clinical manifestations observed in an animal which has beeninfected with a virulent influenza A virus. Examples of such clinicalmanifestations include, but are not limited to, fever, sneezing,coughing, nasal discharge, rates, anorexia and depression. In addition,the phrase “disease caused by an influenza A virus” is defined herein toinclude shedding of virulent virus by the infected animal. Verificationthat clinical manifestations observed in an animal correlate withinfection by virulent equine influenza virus may be made by severalmethods, including the detection of a specific antibody and/or T-cellresponses to equine influenza virus in the animal. Preferably,verification that clinical manifestations observed in an animalcorrelate with infection by a virulent influenza A virus is made by theisolation of the virus from the afflicted animal, for example, byswabbing the nasopharyngeal cavity of that animal for virus-containingsecretions. Verification of virus isolation may be made by the detectionof CPE in tissue culture cells inoculated with the isolated secretions,by inoculation of the isolated secretions into embryonated chicken eggs,where virus replication is detected by the ability of AF from theinoculated eggs to agglutinate erythrocytes, suggesting the presence ofthe influenza virus hemagglutinin protein, or by use of a commerciallyavailable diagnostic test, for example, the Directigen® FLU A test.

As used herein, the term “to protect” includes, for example, to preventor to treat influenza A virus infection in the subject animal. As such,a therapeutic composition of the present invention can be used, forexample, as a prophylactic vaccine to protect a subject animal frominfluenza disease by administering the therapeutic composition to thatanimal at some time prior to that animal's exposure to the virulentvirus.

A therapeutic composition of the present invention, comprising acold-adapted equine influenza virus having a dominant interferencephenotype, can also be used to treat an animal that has been recentlyinfected with virulent influenza A virus or is likely to be subsequentlyexposed in a few days, such that the therapeutic composition immediatelyinterferes with the growth of the virulent virus, prior to the animal'sproduction of antibodies to the virulent virus. A therapeuticcomposition comprising a cold-adapted equine influenza virus having adominant interference phenotype may be effectively administered prior tosubsequent exposure for a length of time corresponding to theapproximate length of time that a cold-adapted equine influenza virus ofthe present invention will repliate in the upper respiratory tract of atreated animal, for example, up to about seven days. A therapeuticcomposition comprising a cold-adapted equine influenza virus having adominant interference phenotype may be effectively administeredfollowing exposure to virulent equine influenza virus for a length oftime corresponding to the time required for an infected animal to showdisease symptoms, for example, up to about two days.

Therapeutic compositions of the present invention can be administered toany animal susceptible to influenza virus disease, for example, humans,swine, horses and other equids, aquatic birds, domestic and game fowl,seals, mink, and whales. Preferably, a therapeutic composition of thepresent invention is administered equids. Even more preferably, atherapeutic composition of the present invention is administered to ahorse, to protect against equine influenza disease.

Current vaccines available to protect horses against equine influenzavirus disease are not effective in protecting young foals, most likelybecause they cannot overcome the maternal antibody present in theseyoung animals, and often, vaccination at an early age, for example 3months of age, can lead to tolerance rather than immunity. In oneembodiment, and in contrast to existing equine influenza virus vaccines,a therapeutic composition comprising a cold-adapted equine influenzavirus of the present invention apparently can produce immunity in younganimals. As such, a therapeutic composition of the present invention canbe safely and effectively administered to young foals, as young as about3 months of age, to protect against equine influenza disease without theinduction of tolerance.

In one embodiment, a therapeutic composition of the present inventioncan be multivalent. For example, it can protect an animal from more thanone strain of influenza A virus by providing a combination of one ormore cold-adapted equine influenza viruses of the present invention, oneor more reassortant influenza A viruses, and/or one or moregenetically-engineered equine influenza viruses of the presentinvention. Multivalent therapeutic compositions can include at least twocold-adapted equine influenza viruses, e.g., against North Americansubtype-2 virus isolates such as A/equine/Kentucky/1/91 (H1N8), andEurasian subtype-2 virus isolates such as A/equine/Suffolk/89 (H3N8); orone or more subtype-2 virus isolates and a subtype-1 virus isolate suchas A/equine/Prague/1/56 (H7N7). Similarly, a multivalent therapeuticcomposition of the present invention can include a cold-adapted equineinfluenza virus and a reassortant influenza A virus of the presentinvention, or two reassortant influenza A viruses of the presentinvention. A multivalent therapeutic composition of the presentinvention can also contain one or more formulations to protect againstone or more other infectious agents in addition to influenza A virus.Such other infectious agents include, but not limited to: viruses;bacteria; fungi and fungal-related microorganisms; and parasites.Preferable multivalent therapeutic compositions include, but are notlimited to, a cold-adapted equine influenza virus, reassortant influenzaA virus, or genetically-engineered equine influenza virus of the presentinvention plus one or more compositions protective against one or moreother infectious agents that afflict horses. Suitable infectious agentsto protect against include, but are not limited to, equine infectiousanemia virus, equine herpes virus, eastern, western, or Venezuelanequine encephalitis virus, tetanus, Streptococcus equi, and Ehrlichiaresticii.

A therapeutic composition of the present invention can be formulated inan excipient that the animal to be treated can tolerate. Examples ofsuch excipients include water, saline, Ringer's solution, dextrosesolution, Hank's solution, and other aqueous physiologically balancedsalt solutions. Excipients can also contain minor amounts of additives,such as substances that enhance isotonicity and chemical or biologicalstability. Examples of buffers include phosphate buffer, bicarbonatebuffer, and Tris buffer, while examples of stabilizers include A1/A2stabilizer, available from Diamond Animal Health, Des Moines, Iowa.Standard formulations can either be liquids or solids which can be takenup in a suitable liquid as a suspension or solution for administrationto an animal. In one embodiment, a non-liquid formulation may comprisethe excipient salts, buffers, stabilizers, etc., to which sterile wateror saline can be added prior to administration.

A therapeutic composition of the present invention may also include oneor more adjuvants or carriers. Adjuvants are typically substances thatenhance the immune response of an animal to a specific antigen, andcarriers include those compounds that increase the half-life of atherapeutic composition in the treated animal. One advantage of atherapeutic composition comprising a cold-adapted equine influenza virusor a reassortant influenza A virus of the present invention is thatadjuvants and carriers are not required to produce an efficaciousvaccine. Furthermore, in many cases known to those skilled in the art,the advantages of a therapeutic composition of the present inventionwould be hindered by the use of some adjuvants or carriers. However, itshould be noted that use of adjuvants or carriers is not precluded bythe present invention.

Therapeutic compositions of the present invention include an amount of acold-adapted equine influenza virus that is sufficient to protect ananimal from challenge with virulent equine influenza virus. In oneembodiment, a therapeutic composition of the present invention caninclude an amount of a cold-adapted equine influenza virus ranging fromabout 10⁵ tissue culture infectious dose-50 (TCID₅₀) units of virus toabout 10⁸ TCID₅₀ units of virus. As used herein, a “TCID₅₀ unit” isamount of a virus which results in cytopathic effect in 50% of thosecell cultures infected. Methods to measure and calculate TCID₅₀ areknown to those skilled in the art and are available, for example, inReed and Muench, 1938, Am. J. of Hyg. 27, 493-497. A preferredtherapeutic composition of the present invention comprises from about10⁶ TCID₅₀ units to about 10⁷ TCID₅₀ units of a cold-adapted equineinfluenza virus or reassortant influenza A virus of the presentinvention. Even more preferred is a therapeutic composition comprisingabout 2×10⁶ TCID₅₀ units of a cold-adapted equine influenza virus orreassortant influenza A virus of the present invention.

The present invention also includes methods to protect an animal againstdisease caused by an influenza A virus comprising administering to theanimal a therapeutic composition of the present invention. Preferred arethose methods which protect an equid against disease caused by equineinfluenza virus, where those methods comprise administering to the equida cold-adapted equine influenza virus. Acceptable protocols toadminister therapeutic compositions in an effective manner includeindividual dose size, number of doses, frequency of dose administration,and mode of administration. Determination of such protocols can beaccomplished by those skilled in the art, and examples are disclosedherein.

A preferable method to protect an animal against disease caused by aninfluenza A virus includes administering to that animal a single dose ofa therapeutic composition comprising a cold-adapted equine influenzavirus, a reassortant influenza A virus, or genetically-engineered equineinfluenza virus of the present invention. A suitable single dose is adose that is capable of protecting an animal from disease whenadministered one or more times over a suitable time period. The methodof the present invention may also include administering subsequent, orbooster doses of a therapeutic composition. Booster administrations canbe given from about 2 weeks to several years after the originaladministration. Booster administrations preferably are administered whenthe immune response of the animal becomes insufficient to protect theanimal from disease. Examples of suitable and preferred dosage schedulesare disclosed in the Examples section.

A therapeutic composition of the present invention can be administeredto an animal by a variety of means, such that the virus will enter andreplicate in the mucosal cells in the upper respiratory tract of thetreated animal. Such means include, but are not limited to, intranasaladministration, oral administration, and intraocular administration.Since influenza viruses naturally infect the mucosa of the upperrespiratory tract, a preferred method to administer a therapeuticcomposition of the present invention is by intranasal administration.Such administration may be accomplished by use of a syringe fitted withcannula, or by use of a nebulizer fitted over the nose and mouth of theanimal to be vaccinated.

The efficacy of a therapeutic composition of the present invention toprotect an animal against disease caused by influenza A virus can betested in a variety of ways including, but not limited to, detection ofantibodies by, for example, hemagglutination inhibition (HAI) tests,detection of cellular immunity within the treated animal, or challengeof the treated animal with virulent equine influenza virus to determinewhether the treated animal is resistant to the development of disease.In addition, efficacy of a therapeutic composition of the presentinvention comprising a cold-adapted equine influenza virus having adominant interference phenotype to ameliorate or reduce disease symptomsin an animal previously inoculated or susceptible to inoculation with avirulent, wild-type equine influenza virus can be tested by screeningfor the reduction or absence of disease symptoms in the treated animal.

The present invention also includes methods to produce a therapeuticcomposition of the present invention. Suitable and preferred methods formaking a therapeutic composition of the present invention are disclosedherein. Pertinent steps involved in producing one type of therapeuticcomposition of the present invention, i.e., a cold-adapted equineinfluenza virus, include (a) passaging a wild-type equine influenzavirus in vitro, for example, in embryonated chicken eggs; (b) selectingviruses that grow at a reduced temperature; (c) repeating the passagingand selection steps one or more times, at progressively lowertemperatures, until virus populations are selected which stably grow atthe desired lower temperature; and (d) mixing the resulting viruspreparation with suitable excipients.

The pertinent steps involved in producing another type of therapeuticcomposition of the present invention, i.e., a reassortant influenza Avirus having at least one genome segment of an equine influenza virusgenerated by adaptation, includes the steps of (a) mixing the genomesegments of a donor cold-adapted equine influenza virus, whichpreferably also has the phenotypes of attenuation, temperaturesensitivity, or dominant interference, with the genome segments of arecipient influenza A virus, and (b) selecting reassortant viruses thathave at least one identifying phenotype of the donor equine influenzavirus. Identifying phenotypes to select for include attenuation,cold-adaptation, temperature sensitivity, and dominant interference.Methods to screen for these phenotypes are well known to those skilledin the art, and are disclosed herein. It is preferable to screen forviruses that at least have the phenotype of attenuation.

Using this method to generate a reassortant influenza A virus having atleast one genome segment of a equine influenza virus generated bycold-adaptation, one type of reassortant virus to select for is a “6+2”reassortant, where the six “internal gene segments,” i.e., those codingfor the NP, PB2, PB 1, PA, M, and NS genes, are derived from the donorcold-adapted equine influenza virus genome, and the two “external genesegments,” i.e., those coding for the HA and NA genes, are derived fromthe recipient influenza A virus. A resultant virus thus produced canhave the cold-adapted, attenuated, temperature sensitive, and/orinterference phenotypes of the donor cold-adapted equine influenzavirus, but the antigenicity of the recipient strain.

The present invention includes nucleic acid molecules isolated fromequine influenza virus wild type strain A/equine/Kentucky/1/91 (H3N8),and cold-adapted equine influenza virus EIV-P821.

In accordance with the present invention, an isolated nucleic acidmolecule is a nucleic acid molecule that has been removed from itsnatural milieu (i.e., that has been subject to human manipulation) andcan include DNA, RNA, or derivatives of either DNA or RNA. As such,“isolated” does not reflect the extent to which the nucleic acidmolecule has been purified.

The present invention includes nucleic acid molecules encoding wild-typeand cold-adapted equine influenza virus proteins. Nucleic acid moleculesof the present invention can be prepared by methods known to one skilledin the art. Proteins of the present invention can be prepared by methodsknown to one skilled in the art, i.e., recombinant DNA technology.Preferred nucleic acid molecules have coding strands comprising nucleicacid sequences SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:44, SEQ ID NO:46, SEQ IDNO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:53, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:62, SEQ IDNO: 64, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:70, SEQ IDNO:71, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ IDNO:82 , SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:90, SEQ IDNO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:103, SEQ IDNO:105, SEQ ID NO:106 and SEQ ID NO:108 and/or a complement thereofComplements are defined as two single strands of nucleic acid in whichthe nucleotide sequence is such that they will hybridize as a result ofbase pairing throughout their full length. Given a nucleotide sequence,one of ordinary skill in the art can deduce the complement.

Preferred nucleic acid molecules encoding equine influenza M proteinsare nei_(wt)M₁₀₂₃, nei_(wt1)M₁₀₂₃, nei_(wt2)M₁₀₂₃, nei_(wt)M₇₅₆,nei_(wt1)M₇₅₆, nei_(wt2)M₇₅₆, nei_(ca1)M₁₀₂₃, nei_(ca2)M₁₀₂₃,nei_(ca1)M₇₅₆, and/or nei_(ca2)M₇₅₆, the coding strands of which arerepresented by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, and/or SEQ IDNO:6.

Preferred nucleic acid molecules encoding equine influenza HA proteinsare nei_(wt)HA₁₇₆₂, nei_(wt)HA₁₆₉₅, nei_(ca1)HA₁₇₆₂, nei_(ca2)HA₁₇₆₂,nei_(ca1)HA₁₆₉₅, and /or nei_(ca2)HA₁₆₉₅, the coding strands of whichare represented by SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, and/or SEQ IDNO:12.

Preferred nucleic acid molecules encoding equine influenza PB2-Nproteins are nei_(wt)PB2-N₁₂₄₁, nei_(wt)PB2-N₁₂₄₁, nei_(ca1)PB2-N₁₂₄₁nei_(ca2)PB2-N₁₂₄₁, nei_(ca1)PB2-N₁₂₁₄ nei_(ca2), and/or PB2-N₁₂₁₄, thecoding strands of which are represented by SEQ ID NO:13, SEQ ID NO:15,SEQ ID NO:16, and/or SEQ ID NO:18.

Preferred nucleic acid molecules encoding equine influenza PB2-Cproteins are nei_(wt1)PB2-C₁₂₃₃, nei_(wt2)PB2-C₁₂₃₂, nei_(wt)PB2-C₁₁₉₄,nei_(ca1)PB2-C₁₂₃₂, nei_(ca2)PB2-C₁₂₃₁, and/or nei_(ca1)PB2-C₁₁₉₄, thecoding strands of which are represented by SEQ ID NO:19, SEQ ID NO:22,SEQ ID NO:21, SEQ ID NO:23, and/or SEQ ID NO:25.

Preferred nucleic acid molecules encoding equine influenza PB2 proteinsare nei_(wt)PB2₂₃₄₁, nei_(wt)PB2₂₂₇₇, nei_(ca1)PB2₂₃₄₁, and/ornei_(ca1)PB2₂₂₇₇, the coding strands of which are represented by SEQ IDNO:44, SEQ ID NO:46, SEQ ID NO:47, and/or SEQ ID NO:49.

Preferred nucleic acid molecules encoding equine influenza NS proteinsare nei_(wt1)NS₈₉₁, nei_(wt2)NS₈₉₁, nei_(wt1)NS₆₉₀, nei_(wt2)NS₆₉₀,nei_(wt3)NS₈₈₈, nei_(wt3)NS₆₉₀, nei_(wt4)NS₄₆₈, nei_(wt4)NS₂₉₃,nei_(ca1)NS₈₈₈, nei_(ca2)NS₈₈₈, nei_(ca1)NS₆₉₀, and/or nei_(ca2)NS₆₉₀the coding strands of which are represented by SEQ ID NO:50, SEQ IDNO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57 and/or SEQID NO:59.

Preferred nucleic acid molecules encoding equine influenza PB1-Nproteins are nei_(wt1)PB1-N₁₂₂₉, nei_(wt1)PB1N₁₁₉₄, nei_(wt2)PB1-N₆₇₃,nei_(wt2)PB1-N₆₃₆, nei_(ca1)PB1-N₁₂₂₅, nei_(ca1)PB1-N₁₁₈₅,nei_(ca2)PB1-N₁₂₂₁, and/or nei_(ca2)PB1-N₁₁₈₅ the coding strands ofwhich are represented by SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, SEQID NO:67, SEQ ID NO:68, SEQ ID NO:70, and/or SEQ ID NO:71.

Preferred nucleic acid molecules encoding equine influenza PA-C proteinsare nei_(wt1)PA-C₁₂₂₈, nei_(wt1)PA-C₁₁₆₄, nei_(wt2)PA-C₁₂₂₃,nei_(wt2)PA-C₁₁₆₄, nei_(ca1)PA-C₁₂₃₃, nei_(ca2)PA-C₁₂₃₃,nei_(ca1)PA-C₁₁₇₀, and/or nei_(ca2)PA-C₁₁₇₀ the coding strands of whichare represented by SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:79, SEQ IDNO:80, and/or SEQ ID NO:82.

Preferred nucleic acid molecules encoding equine influenza PB1-Cproteins are nei_(wt1)PB1-C₁₂₃₄, nei_(wt1)PB1-C₁₁₈₈, nei_(wt2)PB1-C₁₂₄₀,nei_(wt2)PB1-C₁₁₈₈, nei_(ca1)PB1-C₁₂₄₁, nei_(ca1)PB1-C₁₁₈₈,nei_(ca2)PB1-C₁₂₄₁ and/or nei_(ca2)PB1-C₁₁₈₈, the coding strands ofwhich are represented by SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQID NO:90, SEQ ID NO:91, SEQ I) NO:93, SEQ ID NO:94and/or SEQ ID NO:96.

Preferred nucleic acid molecules encoding equine influenza PB1 proteinsare nei_(wt)PB1₂₃₄₁, nei_(wt)PB1₂₂₇₁, nei_(ca1)PB1₂₃₄₁,nei_(ca1)PB1₂₂₇₁, the coding strands of which are represented by SEQ IDNO:103, SEQ ID NO:105, SEQ ID NO:106, and/or SEQ ID NO:108.

The present invention includes proteins comprising SEQ ID NO:2, SEQ IDNO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ I)NO:20, SEQ ID NO:24, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ IDNO:55, SEQ ID NO:58, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ IDNO:77, SEQ ID NO: 81, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ IDNO:95, SEQ I) NO:104 and SEQ ID NO: 107 as well as nucleic acidmolecules encoding such proteins.

Preferred equine influenza M proteins of the present invention includeproteins encoded by a nucleic acid molecule comprising nei_(wt)M₁₀₂₃,nei_(wt1)M₁₀₂₃, nei_(wt2)M₁₀₂₃, nei_(wt)M₇₅₆, nei_(wt1)M₇₅₆,nei_(wt2)M₇₅₆, nei_(ca1)M₁₀₂₃, nei_(ca2)M₁₀₂₃, nei_(ca1)M₇₅₆, and/ornei_(ca2)M₇₅₆. Preferred equine influenza M proteins are Pei_(wt)M₂₅₂,Pei_(ca1)M₂₅₂, and/or Pei_(ca2)M₂₅₂. In one embodiment, a preferredequine influenza M protein of the present invention is encoded by SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:4, and/or SEQ ID NO:6, and, as such, has anamino acid sequence that includes SEQ ID NO:2 and/or SEQ ID NO:5.

Preferred equine influenza HA proteins of the present invention includeproteins encoded by a nucleic acid molecule comprising nei_(wt)HA₁₇₆₂,nei_(wt)HA₁₆₉₅, nei_(ca1)HA₁₇₆₂, nei_(ca2)HA₁₇₆₂, nei_(ca1)HA₁₆₉₅,and/or nei_(ca2)HA₁₆₉₅. Preferred equine influenza HA proteins are PPei_(wt)HA₅₆₅, Pei_(ca1)HA₅₆₅, and/or Pei_(ca2)HA₅₆₅. In one embodiment,a preferred equine influenza HA protein of the present invention isencoded by SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, and/or SEQ ID NO:12,and, as such, has an amino acid sequence that includes SEQ ID NO:8and/or SEQ ID NO:11.

Preferred equine influenza PB2-N proteins of the present inventioninclude proteins encoded by a nucleic acid molecule comprisingnei_(wt)PB2-N₁₂₄₁, nei_(wt)PB2-N₁₂₁₄, nei_(ca1)PB2-N₁₂₄₁nei_(ca2)PB2-N₁₂₄₁, nei_(ca1)PB2-N₁₂₁₄ nei_(ca2), and/or PB2-N₁₂₁₄.Preferred equine influenza PB2-N proteins are P_(wt)PB2-N₄₀₄,P_(ca1)PB2-N₄₀₄, and/or P_(ca2)PB2-N₄₀₄. In one embodiment, a preferredequine influenza PB2-N protein of the present invention is encoded bySEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, and/or SEQ ID NO:18, and, assuch, has an amino acid sequence that includes SEQ ID NO:14 and/or SEQID NO:17.

Preferred equine influenza PB2-C proteins of the present inventioninclude proteins encoded by a nucleic acid molecule comprisingnei_(wt1)PB2-C₁₂₃₃, nei_(wt2)PB2-C₁₂₃₂, nei_(wt)PB2-C₁₁₉₄,nei_(ca1)PB2-C₁₂₃₂, nei_(ca2)PB2-C₁₂₃₁, and/or nei_(ca1)PB2-C₁₁₉₄.Preferred equine influenza PB2-N proteins are P_(wt)PB2-C₃₉₈,P_(ca1)PB2-C₃₉₈, and/or P_(ca2)PB2-C₃₉₈. In one embodiment, a preferredequine influenza PB2-C protein of the present invention is encoded bySEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:21, SEQ ID NO:23, and/or SEQ IDNO:25, and, as such, has an amino acid sequence that includes SEQ IDNO:20 and/or SEQ ID NO:24.

Preferred equine influenza PB2 proteins of the present invention includeproteins encoded by a nucleic acid molecule comprising nei_(wt)PB2₂₃₄₁,nei_(wt)PB2₂₂₇₇, nei_(ca1)PB2₂₃₄₁, and or nei_(ca1)PB2₂₂₇₇. Preferredequine influenza PB2 proteins are Pei_(wt)PB2₇₅₉,and/or Pei_(ca1)PB2₇₅₉.In one embodiment, a preferred equine influenza PB2 protein of thepresent invention is encoded by SEQ ID NO:44, SEQ ID NO:46, SEQ IDNO:47, and/or SEQ ID NO:49, and, as such, has an amino acid sequencethat includes SEQ ID NO:45 and/or SEQ ID NO:48.

Preferred equine influenza NS proteins of the present invention includeproteins encoded by a nucleic acid molecule comprising nei_(wt1)NS₈₉₁,nei_(wt2)NS₈₉₁, nei_(wt1)NS₆₉₀, nei_(wt3)NS₈₈₈, nei_(wt4)NS₄₆₈,nei_(wt4)NS₂₉₃, nei_(ca1)NS₈₈₈, nei_(ca2)NS₈₈₈, and/or nei_(ca1)NS₆₉₀.Preferred equine influenza NS proteins are Pei_(wt)NS₂₃₀, Pei_(wt4)NS₉₇,and/or Pei_(ca1)NS₂₃₀. In one embodiment, a preferred equine influenzaNS protein of the present invention is encoded by SEQ ID NO:50, SEQ IDNO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57 and/or SEQID NO:59, and, as such, has an amino acid sequence that includes SEQ IDNO:51, SEQ ID NO:55 and/or SEQ ID NO:58.

Preferred equine influenza PB1-N proteins of the present inventioninclude proteins encoded by a nucleic acid molecule comprisingnei_(wt1)PB1-N₁₂₂₉, nei_(wt1)PB1N₁₁₉₄, nei_(wt2)PB1-N₆₇₃,nei_(wt2)PB1-N₆₃₆, nei_(ca1)PB21-N₁₂₂₅, nei_(ca1)PB1-N₁₁₈₅, and/ornei_(ca2)PB1-N₁₂₂₁. Preferred equine influenza PB1-N proteins arePei_(wt1)PB1-N₃₉₈, P_(wt2)PB1-N₂₁₂, and/or P_(ca1)PB1-N₃₉₅. In oneembodiment, a preferred equine influenza PB1-N protein of the presentinvention is encoded by SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, SEQ IDNO:67, SEQ ID NO:68, SEQ ID NO:70, and/or SEQ ID NO:71, and, as such,has an amino acid sequence that includes SEQ ID NO:63, SEQ ID NO:66and/or SEQ ID NO:69.

Preferred equine influenza PB1-C proteins of the present inventioninclude proteins encoded by a nucleic acid molecule comprisingnei_(wt1)PB1-C₁₂₃₄, nei_(wt1)PB1-C₁₁₈₈, nei_(wt2)PB1-C₁₂₄₀,nei_(wt2)PB1-C₁₁₈₈, nei_(ca1)PB1-C₁₂₄₁, nei_(ca1)PB1-C₁₁₈₈,nei_(ca2)PB1-C₁₂₄₁ and/or nei_(ca2)PB1-C₁₁₈₈. Preferred equine influenzaPB1-C proteins are Pei_(wt1)PB1-C₃₉₆, Pei_(wt2)PB1-C₃₉₆Pei_(ca1)PB1-C₃₉₆, and/or Pei_(ca2)PB1-C₃₉₆. In one embodiment, apreferred equine influenza PB1-C protein of the present invention isencoded by SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:90, SEQID NO:91, SEQ ID NO:93, SEQ ID NO:94, and/or SEQ ID NO:96, and, as such,has an amino acid sequence that includes SEQ ID NO:86, SEQ ID NO:89, SEQID NO:92, and/or SEQ ID NO:95.

Preferred equine influenza PB1 proteins of the present invention includeproteins encoded by a nucleic acid molecule comprising nei_(wt)PB1₂₃₄₁,nei_(wt)PB1₂₂₇₁, nei_(ca1)PB1₂₃₄₁, nei_(ca1)PB1₂₂₇₁. Preferred equineinfluenza PB1 proteins are Pei_(wt)PB1₇₅₇, and/or Pei_(ca1)PB1₇₅₇. Inone embodiment, a preferred equine influenza PB1 protein of the presentinvention is encoded by SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:106,and/or SEQ ID NO:108, and, as such, has an amino acid sequence thatincludes SEQ ID NO:104 and/or SEQ ID NO:107.

Preferred equine influenza PA-C proteins of the present inventioninclude proteins encoded by a nucleic acid molecule comprisingnei_(wt1)PA-C₁₂₂₈, nei_(wt1)PA-C₁₁₆₄, nei_(wt2)PA-C₁₂₂₃,nei_(ca1)PA-C₁₂₃₃, nei_(ca2)PA-C₁₂₃₃, and/or nei_(ca1)PA-C₁₁₇₀.Preferred equine influenza PA-C proteins are Pei_(wt1)PA-C₃₈₈, and/orPei_(ca1)PA-C₃₉₀. In one embodiment, a preferred equine influenza PA-Cprotein of the present invention is encoded by SEQ ID NO:76, SEQ IDNO:78, SEQ ID NO:79, SEQ ID NO:80, and/or SEQ ID NO:82, and, as such,has an amino acid sequence that includes SEQ ID NO:77 and/or SEQ IDNO:81.

Nucleic acid sequence SEQ ID NO:1 represents the consensus sequencededuced from the coding strand of PCR amplified nucleic acid moleculesdenoted herein as nei_(wt1)M₁₀₂₃ and nei_(wt2)M₁₀₂₃, the production ofwhich is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:4represents the deduced sequence of the coding strand of PCR amplifiednucleic acid molecules denoted herein as nei_(ca1)M₁₀₂₃ andnei_(ca2)M₁₀₂₃, the production of which is disclosed in the Examples.Nucleic acid sequence SEQ ID NO:7 represents the deduced sequence of thecoding strand of a PCR amplified nucleic acid molecule denoted herein asnei_(wt)HA₁₇₆₂, the production of which is disclosed in the Examples.Nucleic acid sequence SEQ ID NO:10 represents the deduced sequence ofthe coding strand of PCR amplified nucleic acid molecules denoted hereinas nei_(ca1)HA₁₇₆₂ and nei_(ca2)HA₁₇₆₂, the production of which isdisclosed in the Examples. Nucleic acid sequence SEQ ID NO:13 representsthe deduced sequence of the coding strand of a PCR amplified nucleicacid molecule denoted herein as nei_(wt)PB2-N₁₂₄₁, the production ofwhich is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:16represents the deduced sequence of the coding strand of PCR amplifiednucleic acid molecules denoted herein as nei_(ca1)PB2-N₁₂₄₁ andnei_(ca2)PB2-N₁₂₄₁, the production of which is disclosed in theExamples. Nucleic acid sequence SEQ ID NO:19 represents the deducedsequence of the coding strand of a PCR amplified nucleic acid moleculedenoted herein as nei_(wt1)PB2-C₁₂₃₃, the production of which isdisclosed in the examples. Nucleic acid sequence SEQ ID NO:22 representsthe deduced sequence of the coding strand of a PCR amplified nucleicacid molecule denoted herein as nei_(wt2)PB2-C₁₂₃₂, the production ofwhich is disclosed in the examples. Nucleic acid sequence SEQ ID NO:23represents the deduced sequence of the coding strand of a PCR amplifiednucleic acid molecule denoted herein as nei_(ca1)PB2-C₁₂₃₂, theproduction of which is disclosed in the examples. Nucleic acid sequenceSEQ ID NO:44 represents the deduced sequence of the coding strand of aPCR amplified nucleic acid molecule denoted herein as nei_(wt)PB2₂₃₄₁,the production of which is disclosed in the Examples. Nucleic acidsequence SEQ ID NO:47 represents the deduced sequence of the codingstrand of PCR amplified nucleic acid molecules denoted herein asnei_(ca1)PB2₂₃₄₁ the production of which is disclosed in the Examples.Nucleic acid sequence SEQ ID NO:50 represents the deduced sequence ofthe coding strand of a PCR amplified nucleic acid molecule denotedherein as nei_(wt1)NS₈₉₁ and nei_(wt2)NS₈₉₁ the production of which isdisclosed in the examples. Nucleic acid sequence SEQ ID NO:53 representsthe deduced sequence of the coding strand of a PCR amplified nucleicacid molecule denoted herein as nei_(wt3)NS₈₈₈, the production of whichis disclosed in the examples. Nucleic acid sequence SEQ ID NO:54represents the deduced sequence of the coding strand of a PCR amplifiednucleic acid molecule denoted herein as nei_(wt4)NS₄₆₈, the productionof which is disclosed in the examples. Nucleic acid sequence SEQ IDNO:57 represents the deduced sequence of the coding strand of a PCRamplified nucleic acid molecule denoted herein as nei_(ca1)NS888 andnei_(ca1)NS₈₈₇, the production of which is disclosed in the Examples.Nucleic acid sequence SEQ ID NO:62 represents the deduced sequence ofthe coding strand of PCR amplified nucleic acid molecules denoted hereinas nei_(wt1)PB1-N₁₂₂₉, the production of which is disclosed in theExamples. Nucleic acid sequence SEQ ID NO:65 represents the deducedsequence of the coding strand of a PCR amplified nucleic acid moleculedenoted herein as nei_(wt2)PB2-N₆₇₃, the production of which isdisclosed in the examples. Nucleic acid sequence SEQ ID NO:68 representsthe deduced sequence of the coding strand of a PCR amplified nucleicacid molecule denoted herein as nei_(ca1)PB1-N₁₂₂₅, the production ofwhich is disclosed in the examples. Nucleic acid sequence SEQ ID NO:71represents the deduced sequence of the coding strand of a PCR amplifiednucleic acid molecule denoted herein as nei_(ca2)PB1-N₁₂₂₁, theproduction of which is disclosed in the examples. Nucleic acid sequenceSEQ ID NO:76 represents the deduced sequence of the coding strand of aPCR amplified nucleic acid molecule denoted herein as nei_(wt1)PA-C₁₂₂₈,the production of which is disclosed in the examples. Nucleic acidsequence SEQ ID NO:79 represents the deduced sequence of the codingstrand of a PCR amplified nucleic acid molecule denoted herein asnei_(wt2)PA-C₁₂₂₃, the production of which is disclosed in the examples.Nucleic acid sequence SEQ ID NO:80 represents the deduced sequence ofthe coding strand of a PCR amplified nucleic acid molecule denotedherein as nei_(ca1)PA-C₁₂₃₃ and nei_(ca2)PA-C₁₂₃₃ the production ofwhich is disclosed in the examples. Nucleic acid sequence SEQ ID NO:85represents the deduced sequence of the coding strand of a PCR amplifiednucleic acid molecule denoted herein as nei_(ca1)PB1-C₁₂₃₄ theproduction of which is disclosed in the examples. Nucleic acid sequenceSEQ ID NO:88 represents the deduced sequence of the coding strand of aPCR amplified nucleic acid molecule denoted herein as nei_(wt2)PB1-C₁₂₄₀the production of which is disclosed in the examples. Nucleic acidsequence SEQ ID NO:91 represents the deduced sequence of the codingstrand of a PCR amplified nucleic acid molecule denoted herein asnei_(ca1)PB1-C₁₂₄₁ the production of which is disclosed in the examples.Nucleic acid sequence SEQ ID NO:94 represents the deduced sequence ofthe coding strand of a PCR amplified nucleic acid molecule denotedherein as nei_(ca2)PB1-C₁₂₄₁ the production of which is disclosed in theexamples. Nucleic acid sequence SEQ ID NO:103 represents the deducedsequence of the coding strand of a PCR amplified nucleic acid moleculedenoted herein as nei_(wt)PB1₂₃₄₁ the production of which is disclosedin the examples. Nucleic acid sequence SEQ ID NO:105 represents thededuced sequence of the coding strand of a PCR amplified nucleic acidmolecule denoted herein as nei_(wt)PB1₂₂₇₁ the production of which isdisclosed in the examples. Nucleic acid sequence SEQ ID NO:106represents the deduced sequence of the coding strand of a PCR amplifiednucleic acid molecule denoted herein as nei_(ca)PB1₂₃₄₁ the productionof which is disclosed in the examples. Nucleic acid sequence SEQ IDNO:108 represents the deduced sequence of the coding strand of a PCRamplified nucleic acid molecule denoted herein as nei_(ca)PB1₂₂₇₁ theproduction of which is disclosed in the examples. Additional nucleicacid molecules, nucleic acid sequences, proteins and amino acidsequences are described in the Examples.

The present invention includes nucleic acid molecule comprising acold-adapted equine influenza virus encoding an M protein having anamino acid sequence comprising SEQ ID NO:5. Another embodiment of thepresent invention includes a nucleic acid molecule comprising acold-adapted equine influenza virus encoding an HA protein having anamino acid sequence comprising SEQ ID NO:11. Another embodiment of thepresent invention includes a nucleic acid molecule comprising acold-adapted equine influenza virus encoding a PB2-N protein having anamino acid sequence comprising SEQ ID NO:17. Another embodiment of thepresent invention includes a nucleic acid molecule comprising acold-adapted equine influenza virus encoding a PB2-C protein having anamino acid sequence comprising SEQ ID NO:24. Another embodiment of thepresent invention includes a nucleic acid molecule comprising acold-adapted equine influenza virus encoding a PB protein having anamino acid sequence comprising SEQ ID NO:48. Another embodiment of thepresent invention includes a nucleic acid molecule comprising acold-adapted equine influenza virus encoding a NS protein having anamino acid sequence comprising SEQ ID NO:58. Another embodiment of thepresent invention includes a nucleic acid molecule comprising acold-adapted equine influenza virus encoding a PB1-N protein having anamino acid sequence comprising SEQ ID NO:69. Another embodiment of thepresent invention includes a nucleic acid molecule comprising acold-adapted equine influenza virus encoding a PA-C protein having anamino acid sequence comprising SEQ ID NO:81. Another embodiment of thepresent invention includes a nucleic acid molecule comprising acold-adapted equine influenza virus encoding a PB1-C protein having anamino acid sequence comprising SEQ ID NO:92. Another embodiment of thepresent invention includes a nucleic acid molecule comprising acold-adapted equine influenza virus encoding a PB1 protein having anamino acid sequence comprising SEQ ID NO:107.

It should be noted that since nucleic acid sequencing technology is notentirely error-free, the nucleic acid sequences and amino acid sequencespresented herein represent, respectively, apparent nucleic acidsequences of nucleic acid molecules of the present invention andapparent amino acid sequences of M, HA, PB2-N, PB2-C, PB2, NS, PB1-N,PA-C, PB1-C and PB1 proteins of the present invention.

Another embodiment of the present invention is an antibody thatselectively binds to an wild-type virus M, HA, PB2-N, PB2-C, PB2, NS,PB1-N, PA-C, PB1-C and PB1 protein of the present invention. Anotherembodiment of the present invention is an antibody that selectivelybinds to a cold-adapted virus M, HA, PB2-N, PB2-C, PB2, NS, PB1-N, PA-C,PB1-C and PB1 protein of the present invention. Preferred antibodiesselectively bind to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11,SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:45,SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:63,SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:81, SEQ ID NO:86,SEQ ID NO: 89, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:104 and SEQ IDNO:107.

The following examples are provided for the purposes of illustration andare not intended to limit the scope of the present invention.

EXAMPLE 1

This example discloses the production and phenotypic characterization ofseveral cold-adapted equine influenza viruses of the present invention.

A. Parental equine influenza virus, A/equine/Kentucky/1/91 (H3N8)(obtained from Tom Chambers, the University of Kentucky, Lexington, Ky.was subjected to cold-adaptation in a foreign host species, i.e.,embryonated chicken eggs, in the following manner. Embryonated, 10 or11-day old chicken eggs, available, for example, from Truslow Farms,Chestertown, Md. or from HyVac, Adel, Iowa, were inoculated with theparental equine influenza virus by injecting about 0.1 milliliter (ml)undiluted AF containing approximately 10⁶ plaque forming units (pfu) ofvirus into the allantoic cavity through a small hole punched in theshell of the egg. The holes in the eggs were sealed with nail polish andthe eggs were incubated in a humidified incubator set at the appropriatetemperature for three days. Following incubation, the eggs were candledand any non-viable eggs were discarded. AF was harvested from viableembryos by aseptically removing a portion of the egg shell, pullingaside the chorioallantoic membrane (CAM) with sterile forceps andremoving the AF with a sterile pipette. The harvested AF was frozenbetween passages. The AF was then used, either undiluted or diluted1:1000 in phosphate-buffered saline (PBS) as noted in Table 1, toinoculate a new set of eggs for a second passage, and so on. A total of69 passages were completed. Earlier passages were done at either about34° C. (passages 1-2) or about 30° C. and on subsequent passages, theincubation temperature was shifted down either to about 28° C., or toabout 26° C. In order to increase the possibility of the selection ofthe desired phenotype of a stable, attenuated virus, the initial serialpassage was expanded to included five different limbs of the serialpassage tree, A through E, as shown in Table 1.

TABLE 1 Passage history of the limbs A through E. Passage # TemperatureLimb A Limb B Limb C Limb D Limb E 34° C. 1-2 1-2 1-2 1-2 1-2 30° C. 3-8 3-29  3-29  3-29  3-29 28° C.  30-33*  30-68* 30-33 30-69 26° C.  9-65 34-69* 34-65 *the infectious allantoic fluid was diluted 1:1000 inthese passages

B. Virus isolates carried through the cold-adaptation proceduredescribed in section A were tested for temperature sensitivity, i.e., aphenotype in which the cold-adapted virus grows at the lower, orpermissive temperature (e.g., about 34° C.), but no longer forms plaquesat a higher, or non-permissive temperature (e.g., about 37° C. or about39° C.), as follows. At each cold-adaptation passage, the AF was titeredby plaque assay at about 34° C. Periodically, individual plaques fromthe assay were clonally isolated by excision of the plaque area andplacement of the excised agar plug in a 96-well tray containing amonolayer of MDCK cells. The 96-well trays were incubated overnight andthe yield assayed for temperature sensitivity by CPE assay in duplicate96-well trays incubated at about 34° C. and at about 39° C. The percentof the clones that scored as temperature sensitive mutants by thisassay, i.e., the number of viral plaques that grew at 34° C. but did notgrow at 39° C., divided by the total number of plaques, was calculated,and is shown in Table 2. Temperature sensitive isolates were thenevaluated for protein synthesis at the non-permissive temperature byvisualization of radiolabeled virus-synthesized proteins by SDSpolyacrylamide gel electrophoresis (SDS-PAGE).

TABLE 2 Percent of isolated Clones that were temperature sensitive.Percent Temperature Sensitive Passage # Limb A Limb B Limb C Limb D LimbE p36 56% 66% 0% 66% 54% p46 80% 60% 75% p47 80% p48 100% p49 100% 100%50% p50 90% p51 100% p52 57% p62 100% 100% p65 100% p66 100% 88%

From the clonal isolates tested for temperature sensitivity, two wereselected for further study. Clone EIV-P821 was selected from the 49thpassage of limb B and clone EIV-P824 was selected from the 48th passageof limb C, as defined in Table 1. Both of these virus isolates weretemperature sensitive, with plaque formation of both isolates inhibitedat a temperature of about 39° C. At this temperature, protein synthesiswas completely inhibited by EIV-P821, but EIV-P824 exhibited normallevels of protein synthesis. In addition, plaque formation by EIV-P821was inhibited at a temperature of about 37° C., and at this temperature,late gene expression was inhibited, i.e., normal levels of NP proteinwere synthesized, reduced or no M1 or HA proteins were synthesized, andenhanced levels of the polymerase proteins were synthesized. Thephenotype observed at 37° C., being typified by differential viralprotein synthesis, was distinct from the protein synthesis phenotypeseen at about 39° C., which was typified by the inhibition of synthesisof all viral proteins. Virus EIV-P821 has been deposited with theAmerican Type Culture Collection (ATCC) under Accession No. ATCCVR-2625, and virus EIV-P824 has been deposited with the ATCC underAccession No. ATCC VR-2624.

C. Further characterization of the mutations in isolate EIV-P821 werecarried out by reassortment analysis, as follows. Reassortment analysisin influenza viruses allows one skilled in the art, under certaincircumstances, to correlate phenotypes of a given virus with putativemutations occurring on certain of the eight RNA segments that comprisean influenza A virus genome. This technique is described, for example,in Palese, et al., ibid. A mixed infection of EIV-P821 and an avianinfluenza virus, A/mallard/New York/6750/78 was performed as follows.MDCK cells were co-infected with EIV-P821 at a multiplicity of infection(MOI) of 2 pfu/cell and A/mallard/New York/6750/78 at an MOI of either2, 5, or 10 pfu/cell. The infected cells were incubated at a temperatureof about 34° C. The yields of the various co-infections were titered andindividual plaques were isolated at about 34° C., and the resultantclonal isolates were characterized as to whether they were able to growat about 39° C. and about 37° C., and express their genes, i.e.,synthesize viral proteins, at about 39° C., about 37° C., and about 34°C. Protein synthesis was evaluated by SDS-PAGE analysis of radiolabeledinfected-cell lysates. The HA, NP and NS-1 proteins of the two parentviruses, each of which is encoded by a separate genome segment, weredistinguishable by SDS-PAGE analysis, since these particular viralproteins, as derived from either the equine or the avian influenzavirus, migrate at different apparent molecular weights. In this way itwas possible, at least for the HA, NP, and NS-1 genes, to evaluatewhether certain phenotypes of the parent virus, e.g., the temperaturesensitive and the protein synthesis phenotypes, co-segregate with thegenome segments carrying these genes. The results of the reassortmentanalyses investigating co-segregation of a) the mutation inhibitingplaque formation, i.e., the induction of CPE, at a non-permissivetemperature of about 39° C. or b) the mutation inhibiting proteinsynthesis at a non-permissive temperature of about 39° C. with each ofthe EIV-P821 HA, NP and NS-1 proteins are shown in Tables 3 and 4,respectively.

TABLE 3 Reassortment analysis of the EIV-P821 39° C. plaque formationphenotype with avian influenza virus, A/mallard/New York/6750/78 GeneVirus ts+¹ ts−² HA avian 26 13 equine 11 44 NP avian 37 8 equine 0 49NS-1 avian 9 8 equine 12 20 ¹number of clonal isolates able to induceCPE in tissue culture cells at a temperature of about 39° C. ²number ofclonal isolates inhibited in the ability to induce CPE in tissue culturecells at a temperature of about 39° C.

TABLE 4 Reassortment analysis of the EIV-P821 39° C. protein synthesisphenotype with avian influenza virus, A/mallard/New York/6750/78 GeneVirus ts+¹ ts−² HA avian 18 1 equine 11 7 NP avian 34 5 equine 7 8 NS-1avian 10 4 equine 14 5 ¹number of clonal isolates which synthesize allviral proteins at a temperature of about 39° C. ²number of clonalisolates inhibited in the ability to synthesize all viral proteins at atemperature of about 39° C.

The results demonstrated an association of the equine NP gene with amutation causing the inability of EIV-P821 to form plaques at anon-permissive temperature of about 39° C., but the results did notsuggest an association of any of the HA, NP, or NS-1 genes with amutation causing the inability of EIV-P821 to express viral proteins ata non-permissive temperature of about 39° C. Thus, these data alsodemonstrated that the plaque formation phenotype and the proteinsynthesis phenotype observed in virus EIV-P821 were the result ofseparate mutations.

D. Studies were also conducted to determine if cold-adapted equineinfluenza viruses of the present invention have a dominant interferencephenotype, that is, whether they dominate in mixed infection with thewild type parental virus A/Kentucky/1/91 (H3N8). The dominantinterference phenotype of viruses EIV-P821 and EIV-P824 were evaluatedin the following manner. Separate monolayers of MDCK cells were singlyinfected with the parental virus A/Kentucky/1/91 (H3N8) at an MOI of 2,singly infected with either cold-adapted virus EIV-P821 or EIV-P824 atan MOI of 2, or simultaneously doubly infected with both the parentalvirus and one of the cold adapted viruses at an MOI of 2+2, all at atemperature of about 34° C. At 24 hours after infection, the media fromthe cultures were harvested and the virus yields from the variousinfected cells were measured by duplicate plaque assays performed attemperatures of about 34° C. and about 39° C. This assay took advantageof the fact that cold adapted equine influenza viruses EIV-P821 orEIV-P824 are temperature sensitive and are thus unable to form plaquesat a non-permissive temperature of about 39° C., while the parentalvirus is able to form plaques at both temperatures, thus making itpossible to measure the growth of the parental virus in the presence ofthe cold adapted virus. Specifically, the dominant interference effectof the cold adapted virus on the growth of the parental virus wasquantitated by comparing the virus yield at about 39° C. of the cellssingly infected with parental virus to the yield of the parental virusin doubly infected cells. EIV-P821, in mixed infection, was able toreduce the yield of the parental virus by approximately 200 fold, whileEIV-P824, in mixed infection, reduced the yield of the parental virus byapproximately 3200 fold. This assay therefore showed that cold-adaptedequine influenza viruses EIV-P821 and EIV-P824 both exhibit the dominantinterference phenotype.

E. Virus isolate EIV-MSV+5 was derived from EIV-P821, as follows.EIV-P821 was passaged once in eggs, as described above, to produce aMaster Seed Virus isolate, denoted herein as EIV-MSV0. EIV-MSV0 was thensubjected to passage three additional times in eggs, the virus isolatesat the end of each passage being designated EIV-MSV+1, EIV-MSV+2, andEIV-MSV+3, respectively. EIV-MSV+3 was then subjected to two additionalpassages in MDCK cells, as follows. MDCK cells were grown in 150 cm²tissue culture flasks in MEM tissue culture medium with Hanks Salts,containing 10% calf serum. The cells were then washed with sterile PBSand the growth medium was replaced with about 8 ml per flask ofinfection medium (tissue culture medium comprising MEM with Hanks Salts,1 μg/ml TPCK trypsin solution, 0.125% bovine serum albumin (BSA), and 10mM HEPES buffer). MDCK cells were inoculated with AF containing virusEIV-MSV+3 (for the first passage in MDCK cells) or virus stock harvestedfrom EIV-MSV+4 (for the second passage in MDCK cells), and the viruseswere allowed to adsorb for 1 hour at about 34° C. The inoculum wasremoved from the cell monolayers, the cells were washed again with PBS,and about 100 ml of infection medium was added per flask. The infectedcells were incubated at about 34° C. for 24 hours. The virus-infectedMDCK cells were harvested by shaking the flasks vigorously to disruptthe cell monolayer, resulting in virus isolates EIV-MSV+4 (the firstpassage in MDCK cells), and EIV-MSV+5 (the second passage in MDCKcells).

Viruses EIV-MSVO and EIV-MSV+5 were subjected to phenotypic analysis, asdescribed in section B above, to determine their ability to form plaquesand synthesize viral proteins at temperatures of about 34° C., about 37°C., and about 39° C. Both EIV-MSV0 and EIV-MSV+5 formed plaques intissue culture cells at a temperature of about 34° C., and neither virusisolate formed plaques or exhibited detectable viral protein synthesisat a temperature of about 39° C. Virus EIV-MSV0 had a similartemperature sensitive phenotype as EIV-P821 at a temperature of about37° C., i.e., it was inhibited in plaque formation, and late geneexpression was inhibited. However, EIV-MSV+5, unlike its parent virus,EIV-P821, did form plaques in tissue culture at a temperature of about37° C., and at this temperature, the virus synthesized normal amounts ofall proteins. Virus EIV-MSV+5 has been deposited with the ATCC underAccession No. ATCC VR-2627.

EXAMPLE 2

Therapeutic compositions of the present invention were produced asfollows.

A. A large stock of EIV-P821 was propagated in eggs as follows. About 60specific pathogen-free embryonated chicken eggs were candled andnon-viable eggs were discarded. Stock virus was diluted to about 1.0×10⁵pfu/ml in sterile PBS. Virus was inoculated into the allantoic cavity ofthe eggs as described in Example 1A. After a 3-day incubation in ahumidified chamber at a temperature of about 34° C., AF was harvestedfrom the eggs according to the method described in Example 1A. Theharvested AF was mixed with a stabilizer solution, for example A1/A2stabilizer, available from Diamond Animal Health, Des Moines, Iowa, at25% V/V (stabilizer/AF). The harvested AF was batched in a centrifugetube and was clarified by centrifugation for 10 minutes at 1000 rpm inan IEC Centra-7R refrigerated table top centrifuge fitted with aswinging bucket rotor. The clarified fluid was distributed into 1-mlcryovials and was frozen at about −70° C. Virus stocks were titrated onMDCK cells by CPE and plaque assay at about 34° C.

B. A large stock of EIV-P821 was propagated in MDCK cells as follows.MDCK cells were grown in 150 cm² tissue culture flasks in MEM tissueculture medium with Hanks Salts, containing 10% calf serum. The cellswere then washed with sterile PBS and the growth medium was replacedwith about 8 ml per flask of infection medium. The MDCK cells wereinoculated with virus stock at an MOI ranging from about 0.5 pfu percell to about 0.005 pfu per cell, and the viruses were allowed to adsorbfor 1 hour at about 34° C. The inoculum was removed from the cellmonolayers, the cells were washed again with PBS, and about 100 ml ofinfection medium was added per flask. The infected cells were incubatedat about 34° C. for 24 hours. The virus-infected MDCK cells wereharvested by shaking the flasks vigorously to disrupt the cell monolayerand stabilizer solution was added to the flasks at 25% V/V(stabilizer/virus solution). The supernatants were distributedaseptically into cryovials and frozen at −70° C.

C. Therapeutic compositions comprising certain cold-adapted temperaturesensitive equine influenza viruses of the present invention wereformulated as follows. Just prior to vaccination procedures, such asthose described in Examples 3-7 below, stock vials of EIV-P821 orEIV-MSV+5 were thawed and were diluted in an excipient comprising eitherwater, PBS, or in MEM tissue culture medium with Hanks Salts, containing0.125% bovine serum albumin (BSA-MEM solution) to the desired dilutionfor administration to animals. The vaccine compositions were held on iceprior to vaccinations. All therapeutic compositions were titered on MDCKcells by standard methods just prior to vaccinations and whereverpossible, an amount of the composition, treated identically to thoseadministered to the animals, was titered after the vaccinations toensure that the virus remained viable during the procedures.

EXAMPLE 3

A therapeutic composition comprising cold-adapted equine influenza virusEIV-P821 was tested for safety and its ability to replicate in threehorses showing detectable prior immunity to equine influenza virus asfollows. EIV-P821, produced as described in Example 1A, was grown ineggs as described in Example 2A and was formulated into a therapeuticcomposition comprising 10⁷ pfu EIV-P821/2 ml BSA-MEM solution asdescribed in Example 2C.

Three ponies having prior detectable hemagglutination inhibition (HAI)titers to equine influenza virus were inoculated with a therapeuticcomposition comprising EIV-P821 by the following method. Each pony wasgiven a 2-ml dose of EIV-P821, administered intranasally using a syringefitted with a blunt cannula long enough to reach past the false nostril,1 ml per nostril.

The ponies were observed for approximately 30 minutes immediatelyfollowing and at approximately four hours after vaccination forimmediate type allergic reactions such as sneezing, salivation, laboredor irregular breathing, shaking, anaphylaxis, or fever. The animals werefurther monitored on days 1-11 post-vaccination for delayed typeallergic reactions, such as lethargy or anorexia. None of the threeponies in this study exhibited any allergic reactions from thevaccination.

The ponies were observed daily, at approximately the same time each day,starting two days before vaccination and continuing through day 11following vaccination for clinical signs consistent with equineinfluenza. The ponies were observed for nasal discharge, oculardischarge, anorexia, disposition, heart rate, capillary refill time,respiratory rate, dyspnea, coughing, lung sounds, presence of toxic lineon upper gum, and body temperature. In addition submandibular andparietal lymph nodes were palpated and any abnormalities were described.None of the three ponies in this study exhibited any abnormal reactionsor overt clinical signs during the observation period.

To test for viral shedding in the animals, on days 0 through 11following vaccination, nasopharyngeal swabs were collected from theponies as described in Chambers, et al., 1995, Equine Practice, 17,19-23. Chambers, et al., ibid. Briefly, two sterile Dacron polyestertipped applicators (available, e.g., from Hardwood Products Co.,Guilford, Me.) were inserted, together, into each nostril of the ponies.The swabs (four total, two for each nostril) were broken off into a15-ml conical centrifuge tube containing 2.5 ml of chilled transportmedium comprising 5% glycerol, penicillin, streptomycin, neomycin, andgentamycin in PBS at physiological pH. Keeping the samples on wet ice,the swabs were aseptically wrung out into the medium and thenasopharyngeal samples were divided into two aliquots. One aliquot wasused to attempt isolation of EIV by inoculation of embryonated eggs,using the method described in Example 1. The AF of the inoculated eggswas then tested for its ability to cause hemagglutination, by standardmethods, indicating the presence of equine influenza virus in the AF. Ondays 2 and 3 post-vaccination, the other aliquots were tested for virusby the Directigen® Flu A test, available from Becton-Dickinson(Cockeysville, Md.).

Attempts to isolate EIV from the nasopharyngeal secretions of the threeanimals by egg inoculation were unsuccessful. However on days 2 and 3,all animals tested positive for the presence of virus shedding using theDirectigen Flu A test, consistent with the hypothesis that EIV-P821 wasreplicating in the seropositive ponies.

To test the antibody titers to EIV in the inoculated animals describedin this example, as well as in the animals described in Examples 4-7,blood was collected from the animals prior to vaccination and ondesignated days post-vaccination. Serum was isolated and was treatedeither with trypsin/periodate or kaolin to block the nonspecificinhibitors of hemagglutination present in normal sera. Serum sampleswere tested for hemagglutination inhibition (HAI) titers against arecent EIV isolate by standard methods, described, for example in the“Supplemental assay method for conducting the hemagglutinationinhibition assay for equine influenza virus antibody” (SAM 124),provided by the U.S.D.A. National Veterinary Services Laboratory under 9CFR 113.2.

The HAI titers of the three ponies are shown in Table 5. As can be seen,regardless of the initial titer, the serum HAI titers increased at leastfour-fold in all three animals after vaccination with EIV-P821.

These data demonstrate that cold-adapted equine influenza virus EIV-P821is safe and non-reactogenic in sero-positive ponies, and that theseanimals exhibited an increase in antibody titer to equine influenzavirus, even though they had prior demonstrable titers.

TABLE 5 HAI titers of vaccinated animals* Animal HAI Titer (days aftervaccination) 1D 0 7 14 21 18 40 80 160 160 19 10 20 40 80 25 20 40 32080 *HAI titers are expressed as the reciprocal of the highest dilutionof serum which inhibited hemagglutination of erythrocytes by a recentisolate of equine influenza virus.

EXAMPLE 4

This Example discloses an animal study to evaluate the safety andefficacy of a therapeutic composition comprising cold-adapted equineinfluenza virus EIV-P821.

A therapeutic composition comprising cold-adapted equine influenza virusEIV-P821 was tested for attenuation, as well as its ability to protecthorses from challenge with virulent equine influenza virus, as follows.EIV-P821, produced as described in Example 1, was grown in eggs asdescribed in Example 2A and was formulated into a therapeuticcomposition comprising 10⁷ pfu of virus/2 ml water, as described inExample 2C. Eight EIV-seronegative ponies were used in this study. Threeof the eight ponies were vaccinated with a 2-ml dose comprising 10⁷ pfuof the EIV-P821 therapeutic composition, administered intranasally,using methods similar to those described in Example 3. One pony wasgiven 10⁷ pfu of the EIV-P821 therapeutic composition, administeredorally, by injecting 6 ml of virus into the pharynx, using a 10-mlsyringe which was adapted to create a fine spray by the followingmethod. The protruding “seat” for the attachment of needles was sealedoff using modeling clay and its cap was left in place. About 10 holeswere punched through the bottom of the syringe, i.e., surrounding the“seat,” using a 25-gauge needle. The syringe was placed into theinterdental space and the virus was forcefully injected into the back ofthe mouth. The remaining four ponies were held as non-vaccinatedcontrols.

The vaccinated ponies were observed for approximately 30 minutesimmediately following and at approximately four hours after vaccinationfor immediate type allergic reactions, and the animals were furthermonitored on days 1-11 post-vaccination for delayed type allergicreactions, both as described in Example 3. None of the four vaccinatedponies in this study exhibited any abnormal reactions from thevaccination.

The ponies were observed daily, at approximately the same time each day,starting two days before virus vaccination and continuing through day 11following vaccination for clinical signs, such as those described inExample 3. None of the four vaccinated ponies in this study exhibitedany clinical signs during the observation period. This resultdemonstrated that cold-adapted equine influenza virus EIV-P821 exhibitsthe phenotype of attenuation.

To test for viral shedding in the vaccinated animals, on days 0 through11 following vaccination, nasopharyngeal swabs were collected from theponies as described in Example 3. The nasopharyngeal samples were testedfor virus in embryonated chicken eggs according to the method describedin Example 3.

As shown in Table 6, virus was isolated from only one vaccinated animalusing the egg method. However, as noted in Example 3, the lack ofisolation by this method does not preclude the fact that virusreplication is taking place, since replication may be detected by moresensitive methods, e.g., the Directigen Flu A test.

TABLE 6 Virus isolation in eggs after vaccination. Animal VirusIsolation (days after vaccination) ID Route 0 1 2 3 4 5 6 7 8 9 10 11 91IN − − + + + + + + + + + − 666 IN − − − − − − − − − − − − 673 IN − − − −− − − − − − − − 674 Oral − − − − − − − − − − − −

To test the antibody titers to equine influenza virus in the vaccinatedanimals, blood was collected from the animals prior to vaccination andon days 7, 14, 21, and 28 post-vaccination. Serum samples were isolatedand were tested for hemagglutination inhibition (HAI) titers against arecent EIV isolate according to the methods described in Example 3.

The HAI titers of the four vaccinated ponies are shown in Table 7.

TABLE 7 HAI titers after vaccination. Animal HAI Titer (days aftervaccination) ID Route 0 7 14 21 28 91 IN <10 <10 <10 <10 <10 666 IN 1010 10 20 20 673 IN 10 10 10 20 20 674 Oral 20 40 40 40 40

Unlike the increase in HAI titer observed with the three animalsdescribed in the study in Example 3, the animals in this study did notexhibit a significant increase, i.e., greater than four-fold, in HAItiter following vaccination with EIV-P821.

Approximately four and one-half months after vaccine virusadministration, all 8 ponies, i.e., the four that were vaccinated andthe four non-vaccinated controls, were challenged by the followingmethod. For each animal, 10⁷ pfu of the virulent equine influenza virusstrain A/equine/Kentucky/1/91 (H3N8) was suspended in 5 ml of water. Amask was connected to a nebulizer, and the mask was placed over theanimal's muzzle, including the nostrils. Five (5) ml was nebulized foreach animal, using settings such that it took 5-10 minutes to deliverthe full 5 ml. Clinical observations, as described in Example 3, wereperformed on all animals three days before challenge and daily for 11days after challenge.

Despite the fact that the vaccinated animals did not exhibit markedincreases in their HAI titers to equine influenza virus, all fourvaccinated animals were protected against equine influenza viruschallenge. None of the vaccinated animals showed overt clinical signs orfever, although one of the animals had a minor wheeze for two days. Onthe other hand, all four non-vaccinated ponies shed virus and developedclinical signs and fever typical of equine influenza virus infection.Thus, this example demonstrates that a therapeutic composition of thepresent invention can protect horses from equine influenza disease.

EXAMPLE 5

This Example discloses an additional animal study to evaluateattenuation of a therapeutic composition comprising cold-adapted equineinfluenza virus EIV-P821, and its ability to protect vaccinated horsesfrom subsequent challenge with virulent equine influenza virus.Furthermore, this study evaluated the effect of exercise stress on thesafety and efficacy of the therapeutic composition.

A therapeutic composition comprising cold-adapted equine influenza virusEIV-P821 was tested for safety and efficacy in horses, as follows.EIV-P821, produced as described in Example 1, was grown in eggs asdescribed in Example 2A and was formulated into a therapeuticcomposition comprising 10⁷ pfu virus/5 ml water, as described in Example2C. Fifteen ponies were used in this study. The ponies were randomlyassigned to three groups of five animals each, as shown in Table 8,there being two vaccinated groups and one unvaccinated control group.The ponies in group 2 were exercise stressed before vaccination, whilethe ponies in vaccinate group 1 were held in a stall.

TABLE 8 Vaccination/challenge protocol. Group No. Ponies ExerciseVaccine Challenge 1 5 — Day 0 Day 90 2 5 Days −4 to 0 Day 0 Day 90 3 5 —— Day 90

The ponies in group 2 were subjected to exercise stress on a treadmillprior to vaccination, as follows. The ponies were acclimated to the useof the treadmill by 6 hours of treadmill use at a walk only. The actualexercise stress involved a daily exercise regimen starting 4 days beforeand ending on the day of vaccination (immediately prior to vaccination).The treadmill exercise regimen is shown in Table 9.

TABLE 9 Exercise regimen for the ponies in Group 2. Speed (m/sec) Time(min.) Incline (°) 1.5 2 0 3.5 2 0 3.5 2 7 4.5† 2 7 5.5† 2 7 6.5† 2 77.5† 2 7 8.5† 2 7 3.5 2 7 1.5 10  0† †Speed, in meters per second(m/sec) was increased for each animal every 2 minutes until the heartrate reached and maintained ≧200 beats per minute

Groups 1 and 2 were given a therapeutic composition comprising 10⁷ pfuof EIV-P821, by the nebulization method described for the challengedescribed in Example 4. None of the vaccinated ponies in this studyexhibited any immediate or delayed allergic reactions from thevaccination.

The ponies were observed daily, at approximately the same time each day,starting two days before vaccination and continuing through day 11following vaccination for clinical signs, such as those described inExample 3. None of the vaccinated ponies in this study exhibited anyovert clinical signs during the observation period.

To test for viral shedding in the vaccinated animals, before vaccinationand on days 1 through 11 following vaccination, nasopharyngeal swabswere collected from the ponies as described in Example 3. Thenasopharyngeal samples were tested for virus in embryonated chicken eggsaccording to the method described in Example 3. Virus was isolated fromthe vaccinated animals, i.e., Groups 1 and 2, as shown in Table 10.

TABLE 10 Virus isolation after vaccination. Animal Virus Isolation (daysafter vaccination) Group ID Exercise 0 1 2 3 4 5 6 7 8 9 10 11 1 12 No −− + + + + + − + + − − 16 − − + + + + + − − − − − 17 − − + + + + + + +− + − 165 − − − − − − − − − − − − 688 − − − − − + − + − − − − 2 7 Yes −− − + + + + − − − − − 44 − − − − − − − − − − − − 435 − − + + + + − − − −− − 907 − − − + − + + − − − − − 968 − − − − − + − + − − − −

To test the antibody titers to equine influenza virus in the vaccinatedanimals, blood was collected prior to vaccination and on days 7, 14, 21,and 28 post-vaccination. Serum samples were isolated and were tested forHAI titers against a recent EIV isolate according to the methodsdescribed in Example 3. These titers are shown in Table 11.

TABLE 11 HAI titers after vaccination and after challenge on day 90.Animal Day Post-vaccination Group ID −1 7 14 21 28 91 105 112 119 126 112 <10 <10 <10 <10 <10 <10 80 320 320 640 1 16 <10 <10 20 20 <10 <10 20160 320 320 1 17 <10 <10 10 10 10 10 80 160 160 160 1 165 <10 <10 10 1010 10 80 80 80 80 1 688 <10 <10 20 20 20 20 20 20 20 40 2 7 <10 <10 1010 <10 <10 20 80 80 40 2 44 <10 <10 20 20 20 10 80 320 320 320 2 435 <10<10 20 20 10 <10 20 80 80 80 2 907 <10 <10 10 10 20 10 10 40 80 80 2 968<10 <10 <10 <10 <10 <10 40 160 160 160 3 2 <10 80 640 640 320 3 56 <1080 320 320 320 3 196 <10 20 160 80 80 3 198 10 40 160 320 320 3 200 <1020 80 80 40 Group Description

On day 90 post vaccination, all 15 ponies were challenged with 10⁷ pfuof equine influenza virus strain A/equine/Kentucky/1/91 (H3N8) by thenebulizer method as described in Example 4. Clinical observations, asdescribed in Example 3, were performed on all animals three days beforechallenge and daily for 11 days after challenge. There were no overtclinical signs observed in any of the vaccinated ponies. Four of thefive non-vaccinated ponies developed fever and clinical signs typical ofequine influenza virus infection.

Thus, this example demonstrates that a therapeutic composition of thepresent invention protects horses against equine influenza disease, evenif the animals are stressed prior to vaccination.

EXAMPLE 6

This Example compared the infectivities of therapeutic compositions ofthe present invention grown in eggs and grown in tissue culture cells.From a production standpoint, there is an advantage to growingtherapeutic compositions of the present invention in tissue culturerather than in embryonated chicken eggs. Equine influenza virus,however, does not grow to as high a titer in cells as in eggs. Inaddition, the hemagglutinin of the virus requires an extracellularproteolytic cleavage by trypsin-like proteases for infectivity. Sinceserum contains trypsin inhibitors, virus grown in cell culture must bepropagated in serum-free medium that contains trypsin in order to beinfectious. It is well known by those skilled in the art that suchconditions are less than optimal for the viability of tissue culturecells. In addition, these growth conditions may select for virus withaltered binding affinity for equine cells, which may affect viralinfectivity since the virus needs to bind efficiently to the animal'snasal mucosa to replicate and to stimulate immunity. Thus, the objectiveof the study disclosed in this example was to evaluate whether theinfectivity of therapeutic compositions of the present invention wasadversely affected by growth for multiple passages in in vitro tissueculture.

EIV-P821, produced as described in Example 1, was grown in eggs asdescribed in Example 2A or in MDCK cells as described in Example 2B. Ineach instance, the virus was passaged five times. EIV-P821 was testedfor its cold-adaptation and temperature sensitive phenotypes after eachpassage. The egg and cell-passaged virus preparations were formulatedinto therapeutic compositions comprising 10⁷ pfu virus/2 ml BSA-MEMsolution, as described in Example 2C, resulting in an egg-grown EIV-P821therapeutic composition and an MDCK cell-grown EIV-P821 therapeuticcomposition, respectively.

Eight ponies were used in this study. Serum from each of the animals wastested for HAI titers to equine influenza virus prior to the study. Theanimals were randomly assigned into one of two groups of four ponieseach. Group A received the egg-grown EIV-P821 therapeutic composition,and Group B received the MDCK-grown EIV-P821 therapeutic composition,prepared as described in Example 2B. The therapeutic compositions wereadministered intranasally by the method described in Example 3.

The ponies were observed daily, at approximately the same time each day,starting two days before vaccination and continuing through day 11following vaccination for allergic reactions or clinical signs asdescribed in Example 3. No allergic reactions or overt clinical signswere observed in any of the animals.

Nasopharyngeal swabs were collected before vaccination and daily for 11days after vaccination. The presence of virus material in the nasalswabs was determined by the detection of CPE on MDCK cells infected asdescribed in Example 1, or by inoculation into eggs and examination ofthe ability of the infected AF to cause hemagglutination, as describedin Example 3. The material was tested for the presence of virus only andnot for titer of virus in the sample. Virus isolation results are listedin Table 12. Blood was collected and serum samples from days 0, 7, 14,21 and 28 after vaccination were tested for hemagglutination inhibitionantibody titer against a recent isolate. HAI titers are also listed inTable 12.

TABLE 12 HAI titers and virus isolation after vaccination. HAI Titer(DPV³) Virus Isolation¹ (DPV³) Group² ID 0 7 14 21 28 0 1 2 3 4 5 6 7 89 10 11 1 31 <10 20 160 160 160 — EC — C EC EC C C EC — — — 37 <10 40160 160 160 — EC C C EC C C C — — — — 40 <10 20 80 160 80 — EC EC C — CEC C — EC EC — 41 <10 40 160 160 80 — EC EC C EC C EC EC — — — — 2 32<10 <10 80 80 40 — EC — C — C — C — EC — — 34 <10 20 160 160 160 — EC —C EC C EC C — — — — 35 <10 <10 80 80 40 — EC — C — C — C — EC — — 42 <10<10 80 80 40 — — — C — C EC EC — — — — ¹E = Egg isolation positive; C =CPE isolation positive; — = virus not detected by either of the methods²Group 1: Virus passaged 5× in MDCK cells; Group 2: Virus passaged 5× inEggs ³Days Post-vaccination

The results in Table 12 show that there were no significant differencesin infectivity or immunogenicity between the egg-grown and MDCK-grownEIV-P821 therapeutic compositions.

EXAMPLE 7

This example evaluated the minimum dose of a therapeutic compositioncomprising a cold-adapted equine influenza virus required to protect ahorse from equine influenza virus infection.

The animal studies disclosed in Examples 3-6 indicated that atherapeutic composition of the present invention was efficacious andsafe. In those studies, a dose of 10⁷ pfu, which correlates toapproximately 10⁸ TCID₅₀ units, was used. However, from the standpointsof cost and safety, it is advantageous to use the minimum virus titerthat will protect a horse from disease caused by equine influenza virus.In this study, ponies were vaccinated with four different doses of atherapeutic composition comprising a cold-adapted equine influenza virusto determine the minimum dose which protects a horse against virulentequine influenza virus challenge.

EIV-P821, produced as described in Example 1A, was passaged and grown inMDCK cells as described in Example 2B and was formulated into atherapeutic composition comprising either 2×10⁴, 2×10⁵, 2×10⁶, or 2×10⁷TCID₅₀ units/1 ml BSA-MEM solution as described in Example 2C. Nineteenhorses of various ages and breeds were used for this study. The horseswere assigned to four vaccine groups, one group of three horses andthree groups of four horses, and one control group of four horses (seeTable 13). Each of the ponies in the vaccine groups were given a 1-mldose of the indicated therapeutic composition, administered intranasallyby methods similar to those described in Example 3.

TABLE 13 Vaccination protocol. Vaccine Dose, Group No. No. AnimalsTCID₅₀ Units 1 3 2 × 10⁷ 2 4 2 × 10⁶ 3 4 2 × 10⁵ 4 4 2 × 10⁴ 5 4 control

The ponies were observed for approximately 30 minutes immediatelyfollowing and at approximately four hours after vaccination forimmediate type reactions, and the animals were further monitored on days1-11 post-vaccination for delayed type reactions, both as described inExample 3. None of the vaccinated ponies in this study exhibited anyabnormal reactions or overt clinical signs from the vaccination.

Blood for serum analysis was collected 3 days before vaccination, ondays 7, 14, 21, and 28 after vaccination, and after challenge on Days 35and 42. Serum samples were tested for HAI titers against a recent EIVisolate according to the methods described in Example 3. These titersare shown in Table 14. Prior to challenge on day 29, 2 of the 3 animalsin group 1, 4 of the 4 animals in group 2, 3 of the 4 animals in group3, and 2 of the 4 animals in group 4 showed at least 4-fold increases inHAI titers after vaccination. In addition, 2 of the 4 control horsesalso exhibited increases in HAI titers. One interpretation for thisresult is that the control horses were exposed to vaccine virustransmitted from the vaccinated horses, since all the horses in thisstudy were housed in the same barn.

TABLE 14 HAI titers post-vaccination and post-challenge, and challengeresults. Dose in Chall. TCID₅₀ Animal Vaccination on Day 0, Challenge onDay 29 Sick No. units ID −1 7 14 21 28 35 42 +/− 1 2 × 10⁷ 41 <10 <10 1040 10 20 80 − 42 40 40 40 40 40 <10 80 − 200 <10 <10 80 40 160 40 40 − 22 × 10⁶ 679 <10 10 40 40 40 20 20 − 682 <10 <10 40 40 40 40 40 − 795 2080 160 160 320 320 640 − R <10 10 40 20 160 40 40 − 3 2 × 10⁵ 73 <10 <10160 40 80 160 160 − 712 <10 <10 20 20 40 40 20 − 720 <10 20 80 40 80 80160 − 796 <10 <10 <10 <10 <10 10 80 + 4 2 × 10⁴ 75 <10 <10 <10 <10 <10<10 160 + 724 <10 >10 <10 <10 <10 20 320 + 789 <10 10 320 160 320 320320 − 790 <10 <10 80 40 160 80 40 5 Control 12 <10 <10 <10 20 20 40 40 −22 10 20 40 10 160 40 640 − 71 <10 <10 <10 <10 10 20 160 + 74 <10 <10<10 <10 <10 <10 20 +

On day 29 post vaccination, all 19 ponies were challenged with equineinfluenza virus strain A/equine/Kentucky/1/91 (H3N8) by the nebulizermethod as described in Example 4. The challenge dose was prospectivelycalculated to contain about 10⁸ TCID₅₀ units of challenge virus in avolume of 5 ml for each animal. Clinical observations, as described inExample 3, were monitored beginning two days before challenge, the dayof challenge, and for 11 days following challenge. As shown in Table 14,no animals in groups 1 or 2 exhibited clinical signs indicative ofequine influenza disease, and only one out of four animals in group 3became sick. Two out of four animals in group 4 became sick, and onlytwo of the four control animals became sick. The results in Table 14suggest a correlation between seroconversion and protection fromdisease, since, for example, the two control animals showing increasedHAI titers during the vaccination period did not show clinical signs ofequine influenza disease following challenge. Another interpretation,however, was that the actual titer of the challenge virus may have beenless than the calculated amount of 10⁸ TCID₅₀ units, since, based onprior results, this level of challenge should have caused disease in allthe control animals.

Nonetheless, the levels of seroconversion and the lack of clinical signsin the groups that received a therapeutic composition comprising atleast 2×10⁶ TCID₅₀ units of a cold-adapted equine influenza virussuggests that this amount was sufficient to protect a horse againstequine influenza disease. Furthermore, a dose of 2×10⁵ TCID₅₀ unitsinduced seroconversion and gave clinical protection from challenge in 3out of 4 horses, and thus even this amount may be sufficient to confersignificant protection in horses against equine influenza disease.

EXAMPLE 8

This example discloses an animal study to evaluate the duration ofimmunity of a therapeutic composition comprising cold-adapted equineinfluenza virus EIV-P821.

A therapeutic composition comprising cold-adapted equine influenza virusEIV-P821, produced as described in Example 1, was grown in eggssimilarly to the procedure described in Example 2A, was expanded bypassage in MDCK cells similarly to the procedure described in Example2B, and was formulated into a therapeutic composition as described inExample 2C. Thirty horses approximately 11 to 12 months of age were usedfor this study. Nineteen of the horses were each vaccinated intranasallyinto one nostril using a syringe with a delivery device tip attached tothe end, with a 1.0 ml dose comprising 6 logs of TCID₅₀ units of theEIV-P821 therapeutic composition. Vaccinations were performed on Day 0.

The horses were observed on Day 0 (before vaccination and up to 4 hourspost-vaccination) and on Study Days 1, 2, 3, 7, 15, and 169post-vaccination. On these days, a distant examination for a period ofat least 15 minutes was performed. This distant examination includedobservation for demeanor, behavior, coughing, sneezing, and nasaldischarge. The examination on Day 169 also served to confirm that thehorses were in a condition of health suitable for transport to thechallenge site which was located approximately 360 miles from thevaccination site.

The animals were acclimated to the challenge site and were observedapproximately daily by a veterinarian or animal technician for evidenceof disease. A general physical examination was performed on Day 171post-vaccination to monitor the following: demeanor, behavior, coughing,sneezing, and nasal discharge. From Days 172 to 177, similarobservations as well as rectal temperature were recorded, according tothe judgment of the attending veterinarian for any individual horse withabnormal clinical presentation.

No vaccinated horses showed any adverse reactions post-vaccination. Onevaccinate was found dead about two months after vaccination. This horseshowed no evidence of adverse reaction when observed for at least onemonth after vaccination. Although no cause of death could be firmlyestablished, the death was not instantaneous and was considered to beconsistent with possible contributing factors such as colic, bonefracture, or severe worm burden. Since there was no other evidence forany adverse reactions post-vaccination in any other vaccinates, it ishighly unlikely that the vaccine contributed to any adverse reaction inthis case.

Challenges were performed on Day 181 post-vaccination. The followingwild-type isolate of equine influenza virus previously shown to causedisease in horses was used as the challenge virus:A/equine/2/Kentucky/91. Prior to infection of each challenge group, thechallenge material was rapidly thawed at approximately 37° C. The viruswas diluted with phosphate-buffered saline to a total volume ofapproximately 21 ml. The diluted material was stored chilled on iceuntil immediately before inoculation. Before inoculation and at the endof nebulization for each challenge group, a sample of diluted challengevirus was collected for pre- and post-inoculation virus titerconfirmation. Vaccinates and controls were randomly assigned to 4challenge groups of 6 horses each and one challenge group of 5 horses sothat each challenge group contained a mixture of 4 vaccinates and 2controls or 3 vaccinates and 2 controls.

Challenge virus in aerosol form was delivered through a tube insertedthrough a small opening centrally in the plastic ceiling with anultrasonic nebulizer (e.g., DeVilbiss Model 099HD, DeVilbiss HealthcareInc., Somerset, Pa.) for a period of approximately 10 minutes. Thehorses remained in the chamber for a further period of approximately 30minutes after the nebulization had been completed (total exposure time,approximately 40 minutes). At that time, the plastic was removed to ventthe chamber, and the horses were released and returned to their pen. Thechallenge procedure was repeated for each group.

All statistical methods in this study were performed using SAS (SASInstitute, Cary, N.C.), and P<0.05 was considered to be statisticallysignificant. Beginning on Day 178 post-vaccination (three days prior tochallenge) through Day 191 (day 10 post-challenge), the horses wereobserved daily by both distant and individual examinations. Rectaltemperatures were measured at these times. Data from day 0 (challengeday) to day 10 were included in the analysis; see Table 15.

TABLE 15 Effect of challenge on daily temperatures (° C.) in vaccinatedand control horses (least squares means). Vaccinated non-vaccinated Daypost challenge (n = 19) (n = 10) P-value 0 100.7 100.8 0.8434 1 100.5100.4 0.7934 2 103.4 104.9 0.0024 3 101.8 103.9 0.0001 4 101.5 103.20.0002 5 101.7 103.8 0.0001 6 101.3 103.6 0.0001 7 100.7 102.3 0.0007 8100.5 101.4 0.0379 9 100.1 100.3 0.7416 10 100.3 100.5 0.7416 pooledSEM* 0.27 0.38 *Standard error of the mean

Table 15 shows that on days 2 through 8, vaccinated horses had lowertemperatures (P<0.05) than the non-vaccinated control horses.

The distant examination consisted of a period of 20 minutes where thefollowing observations were made: coughing, nasal discharge,respiration, and depression. Scoring criteria are shown in Table 16.

TABLE 16 Clinical signs and scoring index. Clinical Sign DescriptionScore Coughing normal during observation period of 15 min 0 coughingonce during observation 1 coughing twice or more during observation 2Nasal discharge normal 0 abnormal, serous 1 abnormal, mucopurulent 2abnormal, profuse 3 Respiration normal 0 abnormal (dyspnea, tachypnea) 1Depression normal 0 depression present^(†) 1 ^(†)Depression was assessedby subjective evaluation of individual animal behavior that included thefollowing: failure to approach food rapidly, general lethargy,inappetence, and anorexia.

Each horse was scored for each of these categories. Additionally,submandibular lymph nodes were palpated to monitor for possiblebacterial infection. In any case where there was a different valuerecorded for a subjective clinical sign score from an observation on thesame day at the distant versus the individual examination, the greaterscore was used in the compilation and analysis of results. For purposesof assessing the health of the horses prior to final disposition,distant examinations were performed at 14, 18, and 21 dayspost-challenge. Data from days 1 through 10 post-challenge were includedin the analysis. These scores were summed on each day for each horse,and the vaccinates and controls were compared using the Wilcoxon ranksums test. In addition, these scores were summed across all days foreach horse, and compared in the same manner. The mean ranks and meanclinical scores are shown in Tables 17 and 18, respectively. Five dayspost-challenge, the mean rank of scores in the vaccinated horses waslower (P<0.05) than in the non-vaccinated control horses; and thiseffect continued on days 6, 7, 8, 9, and 10 (P<0.05). The cumulativerank over the entire test period was also lower (P<0.05) in thevaccinated horses than the non-vaccinated controls.

TABLE 17 Effect of challenge on clinical sign scores in vaccinated andcontrol horses (mean rank). Vaccinated Non-vaccinated (n = 19), (n =10), Day post challenge mean rank* mean rank P-value 0 13.6 17.6 0.18531 16.4 12.4 0.2015 2 15.1 14.9 0.9812 3 13.3 18.3 0.1331 4 13.5 17.90.1721 5 12.4 19.9 0.0237 6 12.7 19.4 0.0425 7 12.1 20.6 0.0074 8 12.619.6 0.0312 9 13.1 18.7 0.0729 10 12.3 20.1 0.0135 total over 11 days11.8 21.2 0.0051 *By Wilcoxon rank sum test.

TABLE 18 Effect of challenge on clinical sign scores in vaccinated andcontrol horses (mean scores). Day post challenge Vaccinated (n = 19)Non-vaccinated (n = 10) 0 1.2 1.6 1 1.5 0.9 2 2.4 2.5 3 3.2 4.1 4 3.44.3 5 3.2 4.7 6 3.4 4.8 7 3.3 4.7 8 3.2 4.5 9 3.2 3.9 10 2.4 3.4

Nasopharyngeal swabs were obtained on the day prior to challenge and ondays 1 to 8 post-challenge, as described in Example 3, and tested forshed virus by cell culture assay. The percent of horses sheddingchallenge virus in each group is shown in Table 19. The percent ofhorses shedding the challenge virus in the vaccinated group was lower(P<0.05) on days 5 and 6 post-challenge than in the non-vaccinatedcontrols. The mean number of days the challenge virus was shed was alsolower (P<0.05) in the vaccinated group as compared to the non-vaccinatedcontrols.

TABLE 19 Percent of horses shedding virus per day post-challenge andmean number of days of shedding per group. Day post challenge Vaccinated(n = 19) Non-vaccinated (n = 10) −1 0 0   1 63.2 90   2 100 100   3 84.2100   4 100 100   5 47.4 88.9*   6 10.5 77.8*   7 5.3 20   8 0 0 averagenumber of 4.1 5.6* days shedding *Within a time point, vaccinatesdifferent from non-vaccinates (P < 0.05) by either Fisher's exact test(percent data) or Wilcoxon rank sums test (days shedding).

The scores from clinical signs relevant to influenza and the objectivetemperature measurements both demonstrated a statistically significantreduction in the group of vaccinates when compared to those from thecontrol group; this is consistent with an interpretation that thevaccine conferred significant protection from disease.

The ability of horses to shed influenza virus post-challenge was alsosignificantly reduced in vaccinates as compared to controls in both theincidence of horses positive for shedding on certain days post-challengeand the mean number of days of shedding per horse. This decreasedshedding by vaccinates is important in that it should serve to reducethe potential for exposure of susceptible animals to the wild-type virusin an outbreak of influenza.

The results of this study are consistent with the interpretation thatthe vaccine safely conferred protection for 6 months from clinicaldisease caused by equine influenza and reduced the potential for thespread of naturally occurring virulent equine influenza virus. While thedegree of protection from disease was not complete (13 out of 19vaccinates were protected, while 10/10 controls were sick), there was aclear reduction in the severity and duration of clinical illness and anoticeable effect on the potential for viral shedding after exposure toa virulent strain of equine influenza. The finding that both vaccinatesand controls were seronegative immediately prior to challenge at 6months post-immunization suggests that immunity mediated by somethingother than serum antibody may be of primary importance in the ability ofthis vaccine to confer measurable and durable protection.

EXAMPLE 9

This Example discloses an animal study to evaluate the ability of atherapeutic composition comprising cold-adapted equine influenza virusEIV-P821 to aid in the prevention of disease following exposure to aheterologous strain of equine influenza virus.

The heterologous strain tested was A/equine/2/Saskatoon/90, describedgenetically as a Eurasian strain (obtained from Hugh Townsend,University of Saskatchewan). Twenty female Percheron horsesapproximately 15 months of age (at the time of vaccination) were usedfor the efficacy study. The horses were assigned to two groups, onegroup of 10 to be vaccinated and another group of 10 to serve asnon-vaccinated controls. On day 0, the vaccinate group was vaccinated inthe manner described in Example 8.

The challenge material, i.e. equine flu strain A/equine/2/Saskatoon/90[H3N8] was prepared similarly to the preparation in Example 8.Vaccinates and controls were randomly assigned to 4 challenge groups of5 horses each such that each challenge group contained a mixture of 2vaccinates and three controls or vice versa. The challenge procedure wassimilar to that described in Example 8. Challenges were performed on Day28 post-vaccination.

Clinical observations were performed for the vaccinates and controls onDay −4 and on Study Days 0 (before vaccination and up to 4 hourspost-vaccination), 1 to 7, 12, 15 to 17, 19 to 23, 25 to 38, and 42. Fordays on which clinical observations were performed during Days −4 to 42,clinical observations including rectal temperature were recordedaccording to the judgment of the attending veterinarian for anyindividual horse with abnormal clinical presentation. Horses were scoredusing the same criteria as in Example 8 (Table 15). Distant examinationswere performed on these days as described in Example 8. On Day 20 andfrom Days 25 to 38, the horses were also observed by both distant andindividual examinations (also performed as described in Example 8).

Rectal temperatures were measured daily beginning 3 days prior tochallenge, and continuing until 10 days post-challenge. Day 0 is the dayrelative to challenge. Data from days 0 through 10 were included in theanalysis. Statistical methods and criteria were identical to those usedin Example 8. On days 2, 5 and 7, vaccinated horses had statisticallysignificant lower body temperatures than the non-vaccinated controlhorses (Table 20).

TABLE 20 Effect of challenge on daily temperatures (° C.) in vaccinatedand control horses (least squares means). Vaccinated Non-vaccinated Daypost challenge (n = 10) (n = 10) P-value 0 99.9 99.8 0.9098 1 100.5100.3 0.4282 2 101.0 102.8 0.000I 3 100.7 100.6 0.7554 4 101.0 101.30.4119 5 100.8 102.1 0.0004 6 100.4 100.4 0.9774 7 100.3 101.1 0.0325 8100.6 100.7 0.8651 9 100.5 100.6 0.8874 10 100.5 100.1 0.2465 Standarderror of the mean = 0.249.

Data from days 1 through 10 post-challenge were included in theanalysis. These scores were summed on each day for each horse, and thevaccinates and controls were compared using the Wilcoxon rank sums test.All statistical methods were performed as described in Example 9. Inaddition, these scores were summed across all days for each horse, andcompared in the same manner. Mean ranks are shown in Table 21.

TABLE 21 Effect of challenge on clinical sign scores in vaccinated andcontrol horses (mean rank). Vaccinated Non-vaccinated Day post challenge(n = 10) (n = 10) P-value 1 8.85 12.15 0.1741 2 8.80 12.20 0.1932 3 8.9012.10 0.2027 4 7.60 13.40 0.0225 5 6.90 14.10 0.0053 6 7.00 14.00 0.00597 6.90 14.10 0.0053 8 7.60 13.40 0.0251 9 6.90 14.10 0.0048 10 6.1014.90 0.0006 total over 10 days 5.70 15.30 0.0003 *By Wilcoxon 2 sampletest.

On day 4 post-challenge, the mean rank of scores in the vaccinatedhorses was lower (P<0.05) than the non-vaccinated control horses, andthis effect continued throughout the remainder of the study (P<0.05).The cumulative rank over the entire test period was also lower in thevaccinated horses than the non-vaccinated controls (P<0.05).

Nasopharyngeal swabs were collected on days 1 and 8 post-challenge, asdescribed in Example 3. The nasal samples were analyzed for the presenceof virus by cell inoculation with virus detection by cytopathogeniceffect (CPE) or by egg inoculation with virus detection byhemagglutination (HA). The cell-culture assay was performed as generallydescribed by Youngner et al., 1994, J. Clin. Microbiol. 32, 750-754.Serially diluted nasal samples were added to wells containing monolayersof Madin Darby Canine Kidney (MDCK) cells. After incubation, wells wereexamined for the presence and degree of cytopathogenic effect. Thequantity of virus in TCID₅₀ units was calculated by the Reed-Muenchtechnique. The egg infectivity assay was performed as described inExample 1. The percent of horses shedding challenge virus for each assayin each group is shown in Tables 22 and 23. The percent of horsesshedding the challenge virus in the vaccinated group was lower (P<0.05)on days 2 through 7 post-challenge by either method. No differences wereseen on days 1 or 8 post-challenge. The number of days the challengevirus was shed was also lower (P<0.05) in the vaccinated group ascompared to the non-vaccinated controls; see Tables 22 and 23.

TABLE 22 Percent of horses shedding virus following challenge - cellculture assay. Day post challenge Vaccinated (n = 10) Non-vaccinated (n= 10) 1 0  0 2 0  70* 3 0  70* 4 20 100* 5 10 100* 6 20 100* 7 0  80* 80  30 average number of 0.5  5.5* days shedding *Within a time point,vaccinates different from non-vaccinates, P < 0.05 by either Fisher'sexact test (percent data) or Wilcoxon 2 sample test (days shedding).

TABLE 23 Percent of horses shedding virus following challenge - egginfectivity assay. Day post challenge Vaccinated (n = 10) Non-vaccinated(n = 10) 1 0  0 2 0 70* 3 10 70* 4 0 90* 5 10 70* 6 20 90* 7 0 50* 8 0 0 average number of 0.4  4.4* days shedding *Within a time point,vaccinates different from non-vaccinates, P < 0.05 by either Fisher'sexact test (percent data) or Wilcoxon 2 sample test (days shedding).

The extent (severity and duration) of clinical signs of influenza amongvaccinates was substantially reduced relative to the controls. Thescores from clinical signs relevant to influenza and the objectivetemperature measurements both demonstrated a statistically significantreduction in the group of vaccinates when compared to those from thecontrol group; indicating that the vaccine conferred significantprotection from disease by the heterologous strain.

The ability of horses to shed influenza virus post-challenge was alsosignificantly reduced in vaccinates as opposed to controls in both theincidence of horses positive for shedding on certain days post-challengeand the mean number of days of shedding per horse. This decreasedshedding by vaccinates is important in that it should serve to reducethe potential for exposure of susceptible animals to the wild-type virusin an outbreak of influenza.

Overall, the results of this study show that the vaccine conferredprotection against a heterologous challenge by a member of the Eurasianlineage of equine influenza virus strains.

EXAMPLE 10

This Example discloses an animal study to evaluate the ability of atherapeutic composition comprising cold-adapted equine influenza virusEIV-P821 to aid in the prevention of disease following exposure to aheterologous strain of equine influenza virus.

The heterologous strain tested was A/equine/2/Kentucky/98[H3N8](obtained from Tom Chambers, University of Kentucky). Eight poniesaged 5 to 7 months were used for this efficacy study. The horses wereassigned to two groups, one group of 4 to be vaccinated and anothergroup of 4 to serve as non-vaccinated controls. Ponies were vaccinatedas described in Example 8, on Day 0.

Clinical observations were performed for the vaccinates on Study Day 0(before vaccination and at 4 hours post-vaccination), as well as on Days1 to 8, 23, 30 to 50, and 57 post-vaccination. Controls were observedclinically on Days 29 to 50 and 57. The observations were performed andscored as described in Example 8.

The challenge material i.e. equine flu strain from Kentucky/98, wasprepared by passing the isolated virus two times in eggs. The inoculumfor each horse was prepared by thawing 0.5 ml of the virus, thendiluting in 4.5 ml of sterile phosphate-buffered saline. The inoculumwas administered by nebulization using a mask for each individual horseon Day 36 post-vaccination.

The clinical observation scores were summed on each day for each horse,and horses were ranked according to the cumulative total score from days1 to 9 post-challenge. Theses results are shown in Table 24.

TABLE 24 Clinical sign observations: total scores, ranked by totalscore. Halter Total Score^(#) Group Identity Days 1 to 9 post-challenge1-Vaccinate 50 0 1-Vaccinate 52 0 1-Vaccinate 55 1 1-Vaccinate 15 22-Control 61 21 2-Control 20 25 2-Control 7 26 2-Control 13 26 ^(#)Totalscores represent the sum of daily scores (where daily scores equal thesum of scores for coughing, nasal discharge, respiration, anddepression) and are ranked from the lowest (least severe) to highest(most severe) scores.

The results of Table 24 show that the scores for vaccinates were between0 and 2, which was significantly lower than the score for controls,which were between 21 and 26.

Rectal temperatures were measured daily beginning 6 days prior tochallenge, and continuing until 9 days post-challenge. Day 0 is the dayrelative to challenge. Data from days 0 through 9 were included in theanalysis. These results are shown in Table 25.

TABLE 25 Effect of Challenge on daily mean temperatures (° C.) invaccinated and control horses. Day post challenge control vaccinatedifference 0 99.7 99.5 0.2 1 100.0 99.6 0.4 2 103.9 100.2 3.7 3 99.899.2 0.6 4 99.6 99.1 0.5 5 99.8 99.3 0.5 6 99.6 99.3 0.3 7 99.3 99.0 0.38 99.7 99.6 0.1 9 99.5 99.1 0.4

The temperatures of the control horses were higher than the temperaturesof the vaccinated horses on all days. The temperature in control horseswas significantly higher on day 2.

Nasopharyngeal swabs were collected on days 1 and 8, post-challenge, asdescribed in Example 3. These samples were tested for shed virus by anegg infectivity assay as described in Example 1. The results of theassay are shown in Table 26.

TABLE 26 Virus shedding post-challenge detected by egg infectivity.Study day No. 35 37 38 39 40 41 42 43 44 days Days post-challengepositive Identity −1 1 2 3 4 5 6 7 8 per Group No. Detection of virus*horse Vaccinates 15 0 2 0 3 3 0 2 1 0 5 50 0 0 0 0 0 1 0 0 0 1 52 0 0 33 2 2 0 0 0 4 55 0 2 3 1 3 0 0 0 0 4 No. horses positive 0 2 2 3 3 2 1 10 per day Controls 07 0 3 3 3 3 3 3 1 0 7 13 0 3 3 3 3 3 3 1 0 7 20 0 23 3 3 3 3 1 0 7 61 0 3 3 3 3 3 3 2 0 7 No. horses positive 0 4 4 4 4 4 44 0 per day *Values refer to the number of eggs testing positive of 3eggs tested per sample. For statistical analysis, a sample wasconsidered positive for virus if at least 1 egg was positive per sample.

The results of Table 26 show that the number of horses positive per daywas higher for the controls than for the vaccinates. Additionally,control horses were positive for more days than vaccinates.

The scores from clinical signs relevant to influenza and the objectivetemperature measurements both demonstrated significant differences inthe group of vaccinates when compared to the control group; this showsthat the vaccine conferred significant protection from disease caused bythe heterologous strain Kentucky/98.

The ability of horses to shed influenza virus post-challenge was alsosignificantly reduced in vaccinates as opposed to controls in the meannumber of days of shedding per horse. This decreased shedding byvaccinates is important in that it should serve to reduce the potentialfor exposure of susceptible animals to the wild-type virus in anoutbreak of influenza.

Overall, the results of this study show that the vaccine safelyconferred protection to a heterologous challenge by a recent andclinically relevant isolate. When the results of this study are viewedin the light of the protection previously demonstrated againstheterologous challenge with a Eurasian strain (Example 9), there isclear evidence to support the assertion that this modified live vaccinecan confer protection against heterologous as well as homologous equineinfluenza infection.

EXAMPLE 11

This example describes the cloning and sequencing of equine influenza M(matrix) protein nucleic acid molecules for wild type and cold-adaptedequine influenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equineinfluenza virus M protein, were produced as follows. A PCR productcontaining an equine M gene was produced by PCR amplification fromequine influenza virus DNA, and primers w584 and w585, designated SEQ IDNO:26, and SEQ ID NO:27, respectively. A nucleic acid molecule of 1023nucleotides, denoted nei_(wt)M₁₀₂₃, with a coding strand having anucleic acid sequence designated SEQ ID NO:1 was produced by further PCRamplification using the above described PCR product as a template andcloned into pCR 2.1®TA cloning vector, available from Invitrogen,Carlsbad, Calif., using standard procedures recommended by themanufacturer. The primers used were the T7 primer, designated by SEQ IDNO:29 and the REV primer, designated by SEQ ID NO:28. Plasmid DNA waspurified using a mini-prep method available from Qiagen, Valencia,Calif. PCR products were prepared for sequencing using a PRISM™ DyeTerminator Cycle Sequencing Ready Reaction kit, a PRISM™ dRhodamineTerminator Cycle Sequencing Ready Reaction kit, or a PRISM™ BigDye™Terminator Cycle Sequencing Ready Reaction kit, all available from PEApplied Biosystems, Foster City, Calif., following the manufacturer'sprotocol. Specific PCR conditions used with the kit were a rapid ramp to95° C., hold for 10 seconds followed by a rapid ramp to 50° C. with a 5second hold then a rapid ramp to 60° C. with a 4 minute hold, repeatingfor 25 cycles. Different sets of primers were used in differentreactions: T7 and REV were used in one reaction; w584 and w585 were usedin a second reaction; and efM-a1, designated SEQ ID NO:31 and efM-s1,designated SEQ ID NO:30 were used in a third reaction. PCR products werepurified by ethanol/magnesium chloride precipitation. Automatedsequencing of DNA samples was performed using an ABI PRISM™ Model 377with XL upgrade DNA Sequencer, available from PE Applied Biosystems.

Translation of SEQ ID NO:1 indicates that nucleic acid moleculenei_(wt)M₁₀₂₃ encodes a full-length equine influenza M protein of about252 amino acids, referred to herein as Pei_(wt)M₂₅₂, having amino acidsequence SEQ ID NO:2, assuming an open reading frame in which theinitiation codon spans from nucleotide 25 through nucleotide 28 of SEQID NO:1 and the termination codon spans from nucleotide 781 throughnucleotide 783 of SEQ ID NO:1. The region encoding Pei_(wt)M₂₅₂,designated nei_(wt)M₇₅₆, and having a coding strand comprisingnucleotides 25 to 780 of SEQ ID NO:1, is represented by SEQ ID NO:3.

SEQ ID NO:1 and SEQ ID NO:3 represent the consensus sequence obtainedfrom two wild type nucleic acid molecules, which differ in onenucleotide. Nucleotide 663 of nei_(wt1)M₁₀₂₃, i.e., nucleotide 649 ofnei_(wt1)M₇₅₆, was adenine, while nucleotide 663 of nei_(wt2)M₁₀₂₃,i.e., nucleotide 649 of nei_(wt2)M₇₅₆, was guanine. Translation of thesesequences does not result in an amino acid change at the correspondingamino acid; both translate to valine at residue 221 in Pei_(wt)M₂₅₂.

B. A nucleic acid molecule of 1023 nucleotides encoding a cold-adaptedequine influenza virus M, denoted nei_(ca1)M₁₀₂₃, with a coding strandhaving a sequence designated SEQ ID NO:4 was produced by further PCRamplification and cloned into the pCR®-Blunt cloning vector availablefrom Invitrogen, using conditions recommended by the manufacturer, andprimers T7 and REV. Plasmid DNA purification and cycle sequencing wereperformed as described in Example 11, part A. Translation of SEQ ID NO:4indicates that nucleic acid molecule nei_(ca1)M₁₀₂₃ encodes afull-length equine influenza M protein of about 252 amino acids,referred to herein as Pei_(ca1)M₂₅₂, having amino acid sequence SEQ IDNO:5, assuming an open reading frame in which the initiation codon spansfrom nucleotide 25 through nucleotide 28 of SEQ ID NO:4 and thetermination codon spans from nucleotide 781 through nucleotide 783 ofSEQ ID NO:4. The region encoding Pei_(ca1)M₂₅₂, designatednei_(ca1)M₇₅₆, and having a coding strand comprising nucleotides 25 to780 of SEQ ID NO:4, is represented by SEQ ID NO:6. PCR amplification ofa second nucleic acid molecule encoding a cold-adapted equine influenzaM protein in the same manner resulted in molecules nei_(ca2)M₁₀₂₃,identical to nei_(ca1)M₁₀₂₃, and nei_(ca2)M₇₅₆, identical tonei_(ca1)M₇₅₆.

C. Comparison of the nucleic acid sequences of the coding strands ofnei_(wt)M₁₀₂₃ (SEQ ID NO:1) and nei_(ca1)M₁₀₂₃ (SEQ ID NO:4) by DNAalignment reveals the following differences: a G to T shift at base 67,a C to T shift at base 527, and a G to C shift at base 886. Comparisonof the amino acid sequences of proteins Pei_(wt)M₂₅₂ (SEQ ID NO:2) andPei_(ca1)M₂₅₂ (SEQ ID NO:5) reveals the following differences: a V to Lshift at amino acid 23 relating to the G to T shift at base 67 in theDNA sequences; and a T to I shift at amino acid 187 relating to the C toT shift at base 527 in the DNA sequences.

EXAMPLE 12

This example describes the cloning and sequencing of equine influenza HA(hemagglutinin) protein nucleic acid molecules for wild type orcold-adapted equine influenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equineinfluenza virus HA proteins were produced as follows. A PCR productcontaining an equine HA gene was produced by PCR amplification fromequine influenza virus DNA and primers w578 and w579, designated SEQ IDNO:32 and SEQ ID NO:33, respectively. A nucleic acid molecule of 1762nucleotides encoding a wild-type HA protein, denoted nei_(wt)HA₁₇₆₂,with a coding strand having a nucleic acid sequence designated SEQ IDNO:7 was produced by further PCR amplification using the above-describedPCR product as a template and cloned into pCR 2.1®TA cloning vector asdescribed in Example 11A. Plasmid DNA was purified and sequenced as inExample 11A, except that primers used in the sequencing kits were eitherT7 and REV in one case, or HA-1, designated SEQ ID NO:34, and HA-2,designated SEQ ID NO:35, in a second case.

Translation of SEQ ID NO:7 indicates that nucleic acid moleculenei_(wt)HA₁₇₆₂ encodes a full-length equine influenza HA protein ofabout 565 amino acids, referred to herein as Pei_(wt)HA₅₆₅, having aminoacid sequence SEQ ID NO:8, assuming an open reading frame in which theinitiation codon spans from nucleotide 30 through nucleotide 33 of SEQID NO:7 and the termination codon spans from nucleotide 1725 throughnucleotide 1727 of SEQ ID NO:7. The region encoding Pei_(wt)HA₅₆₅,designated nei_(wt)HA₁₆₉₅, and having a coding strand comprisingnucleotides 30 to 1724 of SEQ ID NO:7 is represented by SEQ ID NO:9.

B. A nucleic acid molecule of 1762 nucleotides encoding a cold-adaptedequine influenza virus HA protein, denoted nei_(ca1)HA₁₇₆₂, with acoding strand having a sequence designated SEQ ID NO:10 was produced asdescribed in Example 11B. Plasmid DNA purification and cycle sequencingwere performed as described in Example 12, part A.

Translation of SEQ ID NO:10 indicates that nucleic acid moleculenei_(ca1)HA₁₇₆₂ encodes a full-length equine influenza HA protein ofabout 565 amino acids, referred to herein as Pei_(ca1)HA₅₆₅, havingamino acid sequence SEQ ID NO:11, assuming an open reading frame inwhich the initiation codon spans from nucleotide 30 through nucleotide33 of SEQ ID NO:10 and the termination codon spans from nucleotide 1725through nucleotide 1727 of SEQ ID NO:10. The region encodingPei_(ca1)HA₅₆₅, designated nei_(ca1)HA₁₆₉₅, and having a coding strandcomprising nucleotides 30 to 1724 of SEQ ID NO:10, is represented by SEQID NO:12.

PCR amplification of a second nucleic acid molecule encoding acold-adapted equine influenza HA protein in the same manner resulted inmolecules nei_(ca2)HA₁₇₆₂, identical to nei_(ca1)HA₁₇₆₂, andnei_(ca2)HA₁₆₉₅, identical to nei_(ca1)HA₁₆₉₅.

C. Comparison of the nucleic acid sequences of the coding strands ofnei_(wt)HA₁₇₆₂ (SEQ ID NO:7) and nei_(ca1)HA₁₇₆₂ (SEQ ID NO:10) by DNAalignment reveals the following differences: a C to T shift at base 55,a G to A shift at base 499, a G to A shift at base 671, a C to T shiftat base 738, a T to C shift at base 805, a G to A shift at base 1289,and an A to G shift at base 1368. Comparison of the amino acid sequencesof proteins Pei_(wt)HA₅₆₅ (SEQ ID NO:8) and Pei_(ca1)HA₅₆₅ (SEQ IDNO:11) reveals the following differences: a P to L shift at amino acid18 relating to the C to T shift at base 55 in the DNA sequences; a G toE shift at amino acid 166 relating to the G to A shift at base 499 inthe DNA sequences; an R to W shift at amino acid 246 relating to the Cto T shift at base 738 in the DNA sequences; an M to T shift at aminoacid 268 relating to the T to C shift at base 805 in the DNA sequences;a K to E shift at amino acid 456 relating to the A to G shift at base1368 in the DNA sequences. There is no change of the serine (S) atresidue 223 relating to the G to A shift at base 671 in the DNAsequences, nor is there a change of the arginine (R) at residue 429relating to the G to A shift at base 1289 in the DNA sequences.

EXAMPLE 13

This example describes the cloning and sequencing of equine influenzaPB2 protein (RNA-directed RNA polymerase) nucleic acid moleculescorresponding to the N-terminal portion of the protein, for wild type orcold-adapted equine influenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equineinfluenza virus PB2-N proteins were produced as follows. A PCR productcontaining an N-terminal portion of the equine PB2 gene was produced byPCR amplification from equine influenza virus DNA, and primers w570 andw571, designated SEQ ID NO:36 and SEQ ID NO:37, respectively. A nucleicacid molecule of 1241 nucleotides encoding a wild type PB2-N protein,denoted nei_(wt)PB2-N₁₂₄₁, with a coding strand having a nucleic acidsequence designated SEQ ID NO:13 was produced by further PCRamplification using the above described PCR product as a template andcloned as described in Example 11B. Plasmid DNA was purified andsequenced as in Example 11B, except that only T7 and REV primers wereused in the sequencing kits.

Translation of SEQ ID NO:13 indicates that nucleic acid moleculenei_(wt)PB2-N₁₂₄₁ encodes an N-terminal portion of influenza PB2 proteinof about 404 amino acids, referred to herein as P_(wt)PB2-N₄₀₄, havingamino acid sequence SEQ ID NO:14, assuming an open reading frame inwhich the initiation codon spans from nucleotide 28 through nucleotide30 of SEQ ID NO:13, and the last codon spans from nucleotide 1237through nucleotide 1239. The region encoding P_(wt)PB2-N₄₀₄, designatednei_(wt)PB2-N₁₂₁₄, and having a coding strand comprising nucleotides 28to 1239 of SEQ ID NO:13 is represented by SEQ ID NO:15.

B. A nucleic acid molecule of 1239 nucleotides encoding an N-terminalportion of influenza PB2 cold-adapted equine influenza virus PB2-Nprotein, denoted nei_(ca1)PB2-N₁₂₄₁, with a coding strand having asequence designated SEQ ID NO:16 was produced, and sequenced asdescribed in as in Example 12, part A.

Translation of SEQ ID NO:16 indicates that nucleic acid moleculenei_(ca1)PB2-N₁₂₄₁ encodes an N-terminal portion of equine influenzaPB-2 protein of about 404 amino acids, referred to herein asP_(ca1)PB2-N₄₀₄, having amino acid sequence SEQ ID NO:17, assuming anopen reading frame in which the initiation codon spans from nucleotide28 through nucleotide 30 of SEQ ID NO:16, and the last codon spans fromnucleotide 1237 through nucleotide 1239. The region encodingP_(ca1)PB2-N₄₀₄, designated nei_(ca1)PB2-N₁₂₁₄, and having a codingstrand comprising nucleotides 28 to 1239 of SEQ ID NO:16, is representedby SEQ ID NO:18.

PCR amplification of a second nucleic acid molecule encoding acold-adapted equine influenza PB2-N protein in the same manner resultedin molecules nei_(ca2)PB2-N₁₂₄₁, identical to nei_(ca1)PB2-N₁₂₄₁, andnei_(ca2)PB2-N₁₂₁₄, identical to nei_(ca1)PB2-N₁₂₁₄.

C. Comparison of the nucleic acid sequences of the coding strands ofnei_(wt)PB2-N₁₂₄₁ (SEQ ID NO:13) and nei_(ca1)PB2-N₁₂₄₁ (SEQ ID NO:16)by DNA alignment reveals the following difference: a T to C base shiftat base 370. Comparison of the amino acid sequences of proteinsP_(wt)PB2-N₄₀₄ (SEQ ID NO:14) and P_(ca1)PB2-N₄₀₄ (SEQ ID NO:17) revealsthe following difference: a Y to H shift at amino acid 124 relating tothe a T to C shift at base 370 in the DNA sequence.

EXAMPLE 14

This example describes the cloning and sequencing of equine influenzaPB2 protein (RNA-directed RNA polymerase) nucleic acid moleculescorresponding to the C-terminal portion of the protein, for wild type orcold-adapted equine influenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equineinfluenza virus PB2-C proteins were produced as follows. A PCR productcontaining the C-terminal portion of the equine PB2 gene was produced byPCR amplification using from equine influenza virus DNA and primers w572and w573, designated SEQ ID NO:38 and SEQ ID NO:39, respectively. Anucleic acid molecule of 1233 nucleotides encoding a wild type PB2-Cprotein, denoted nei_(wt)PB2-C₁₂₃₃, with a coding strand having anucleic acid sequence designated SEQ ID NO:19 was produced by furtherPCR amplification using the above-described PCR product as a templateand cloned as described in Example 11B. Plasmid DNA was purified andsequenced as in Example 11A, except that different primers were used inthe sequencing kits. T7 and REV were used in one instance; efPB2-a1,designated SEQ ID NO:40 and efPB2-s1, designated SEQ ID NO:41 were usedin another instance, and efPB2-a2, designated SEQ ID NO:42 and efPB2-s2,designated SEQ ID NO:43 were used in another instance.

Translation of SEQ ID NO:19 indicates that nucleic acid moleculenei_(wt1)PB2-C₁₂₃₃ encodes a C-terminal portion of influenza PB2 proteinof about 398 amino acids, referred to herein as P_(wt)PB2-C₃₉₈, havingamino acid sequence SEQ ID NO:20, assuming an open reading frame havinga first codon spans from nucleotide 3 through nucleotide 5 and atermination codon which spans from nucleotide 1197 through nucleotide1199 of SEQ ID NO:19. Because SEQ ID NO:19 is only a partial genesequence, it does not contain an initiation codon. The region encodingP_(wt)PB2-C₃₉₈, designated nei_(wt)PB2-C₁₁₉₄, and having a coding strandcomprising nucleotides 3 to 1196 of SEQ ID NO:19 is represented by SEQID NO:21.

PCR amplification of a second nucleic acid molecule encoding a wild typeequine influenza PB2-N protein in the same manner resulted in a nucleicacid molecule of 1232 nucleotides denoted nei_(wt2)PB2-N₁₂₃₂, with acoding strand with a sequence designated SEQ ID NO:22.nei_(wt2)PB2-N₁₂₃₂ is identical to nei_(wt1) PB2-C₁₂₃₃, expect thatnei_(wt2)PB2-N₁₂₃₂ lacks one nucleotide on the 5′-end. Translation ofSEQ ID NO:22 indicates that nucleic acid molecule nei_(wt1)PB2-C₁₂₃₃also encodes P_(wt)PB2-C₃₉₈ (SEQ ID NO:20), assuming an open readingframe having a first codon which spans from nucleotide 2 throughnucleotide 4 and a termination codon spans from nucleotide 1196 throughnucleotide 1198 of SEQ ID NO:22. Because SEQ ID NO:22 is only a partialgene sequence, it does not contain an initiation codon. The nucleic acidmolecule having a coding strand comprising nucleotides 2 to 1195 of SEQID NO:22, denoted nei_(wt2)PB2-C₁₁₉₄, is identical to SEQ ID NO:21.

B. A nucleic acid molecule of 1232 nucleotides encoding a C-terminalportion of influenza PB2 cold-adapted equine influenza virus protein,denoted nei_(ca1)PB2-C₁₂₃₂, and having a coding strand having a sequencedesignated SEQ ID NO:23 was produced as described in as in Example 14,part A, except that the pCR®-Blunt cloning vector was used.

Translation of SEQ ID NO:23 indicates that nucleic acid moleculenei_(ca1)PB2-C₁₂₃₂ encodes a C-terminal portion of equine influenza PB-2protein of about 398 amino acids, referred to herein as P_(ca1)PB2-C₃₉₈,having amino acid sequence SEQ ID NO:24, assuming an open reading framehaving a first codon which spans from nucleotide 2 through nucleotide 4and a termination codon spans from nucleotide 1196 through nucleotide1198 of SEQ ID NO:23. Because SEQ ID NO:23 is only a partial genesequence, it does not contain an initiation codon. The region encodingP_(ca1)PB2-C₃₉₈, designated nei_(ca1)PB2-C1194, and having a codingstrand comprising nucleotides 2 to 1195 of SEQ ID NO:23, is representedby SEQ ID NO:25. PCR amplification of a second nucleic acid moleculeencoding a cold-adapted equine influenza PB2-C protein in the samemanner resulted in molecules nei_(ca2)PB2-C₁₂₃₁, containing one lessnucleotide at the 3′ end than nei_(ca1)PB2-N₁₂₄₁; andnei_(ca2)PB2-N₁₂₁₄, identical to nei_(ca1)PB2-N₁₂₁₄.

C. Comparison of the nucleic acid sequences of the coding strands ofnei_(wt1)PB2-C₁₂₃₃ (SEQ ID NO:19) and nei_(ca1)PB2-C₁₂₃₂ (SEQ ID NO:23)by DNA alignment reveals the following differences: an A to C base shiftat base 153 of SEQ ID NO:19, and a G to A base shift at base 929 of SEQID NO:19. Comparison of the amino acid sequences of proteinsP_(wt)PB2-C₃₉₈ (SEQ ID NO:20) and P_(ca1)PB2-₃₉₈ (SEQ ID NO:24) revealsthe following difference: a K to Q shift at amino acid 51 when relatingto the an A to C base shift at base 153 in the DNA sequences. There isno amino acid shift resulting from the G to A base shift at base 929.

EXAMPLE 15

This example describes the cloning and sequencing of equine influenzaPB2 protein (RNA-directed RNA polymerase) nucleic acid molecules forwild type or cold-adapted equine influenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equineinfluenza virus PB2 proteins were produced as follows. The wild type orcold-adapted equine influenza genes were cloned in two fragments, theN-terminal portion was produced as in Example 13 and the C-terminalportion of the gene was produced as in Example 14. The DNA sequence forthe wild type equine influenza PB2 gene was generated by combining theconsensus sequences for the wild type PB2-N protein , denotednei_(wt)PB2-N₁₂₄₁ (SEQ ID NO:13) with the gene fragments for the wildtype PB2-C protein, denoted nei_(wt1)PB2-C₁₂₃₃ (SEQ ID NO:19) andnei_(wt2)PB2-C₁₂₃₂ (SEQ ID NO:22). The result of combining the consensussequences from the N-terminal and C-terminal portions of the PB2 wildtype influenza virus yielded a complete DNA sequence denotednei_(wt)PB2₂₃₄₁(SEQ ID NO:44). Translation of SEQ ID NO:44 indicatesthat the nucleic acid molecule nei_(wt)PB2₂₃₄₁ encodes a full lengthequine influenza PB2 protein of about 759 amino acids referred to hereinas Pei_(wt)PB2₇₅₉, having amino acid sequence SEQ ID NO:45, assuming anopen reading frame in which the initiation codon spans from nucleotide28 through nucleotide 30 of SEQ ID NO:44 and the termination codon spansfrom nucleotide 2305 through nucleotide 2307 of SEQ ID NO:44. The regionencoding Pei_(wt)PB2₇₅₉, designated nei_(wt)PB2₂₂₇₇, and having a codingstrand comprising nucleotides 28 to 2304 of SEQ ID NO:44, is SEQ IDNO:46.

B. A DNA sequence of 2341 nucleotides encoding a cold-adapted equineinfluenza virus PB2, denoted nei_(ca1)PB2₂₃₄₁, with a sequence denotedSEQ ID NO:47 was produced by combining the sequences for the N-terminaland C-terminal portions of the PB2 cold-adapted equine influenza gene.The clones for the N-terminal sequences are denoted nei_(ca1)PB2-N₁₂₄₁and nei_(ca2)PB2-N₁₂₄₁ which are identical and are represented by SEQ IDNO:16. The clones for the C-terminal sequences are denotednei_(ca1)PB2-C₁₂₃₂ and nei_(ca2)PB2-C₁₂₃₁. represented by SEQ ID NO:23.

Translation of SEQ ID NO:47 indicates that nucleic acid moleculenei_(ca1)PB2₂₃₄₁ encodes a full-length equine influenza PB2 protein ofabout 759 amino acids, referred to herein as Pei_(ca1)PB2₇₅₉, havingamino acid sequence SEQ ID NO:48, assuming an open reading frame inwhich the initiation codon spans from nucleotide 28 through nucleotide30 of SEQ ID NO:47 and the termination codon spans from nucleotide 2305through nucleotide 2307 of SEQ ID NO:47. The region encodingPei_(ca1)PB2₇₅₉ designated nei_(ca1)PB2₂₂₇₇ and having a coding strandcomprising nucleotides 28 to 2304 of SEQ ID NO:49.

C. Comparison of the nucleic acid sequences of the coding strands ofnei_(wt)PB₂₃₄₁ (SEQ ID NO:44) and nei_(ca1)PB2₂₃₄₁ (SEQ ID NO:47) by DNAalignment reveals the following differences: a T to C base shift at base370, a A to C base shift at base 1261, and a G to A base shift at base2037. Comparison of the amino acid sequences of proteins Pei_(wt)PB2₇₅₉(SEQ ID NO:45) and Pei_(ca1)PB2₇₅₉ (SEQ ID NO:48) reveals the followingdifferences: a Y to H shift at amino acid 124 relating to the a T to Cshift at base 370 in the DNA sequence, a K to Q shift at amino acid 421relating to the A to C shift at base 1261 in the DNA sequence. The thirdnucleotide shift at base 2037 does not result in an amino acid shift atamino.

EXAMPLE 16

This example describes the cloning and sequencing of equine influenza NS(nonstructural) protein nucleic acid molecules for wild type orcold-adapted equine influenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equineinfluenza virus NS proteins were produced as follows. A PCR productcontaining an equine NS gene was produced by PCR amplification fromequine influenza virus DNA and primers w586 and w587, designated SEQ IDNO:59 and SEQ ID NO:60, respectively. A nucleic acid molecule of 891nucleotides encoding a wild-type NS protein, denoted nei_(wt)NS₈₉₁, witha coding strand having a nucleic acid sequence designated SEQ ID NO:50was produced by further PCR amplification using the above-described PCRproduct as a template and cloned into pCR 2.1®TA cloning vector asdescribed in Example 11A. Plasmid DNA was purified and sequenced as inExample 11A, except that primers used in the sequencing kits were onlyT7 and REV were used in the sequencing kits.

Translation of SEQ ID NO:50 indicates that nucleic acid moleculenei_(wt1)NS₈₉₁ encodes a full-length equine influenza NS protein ofabout 230 amino acids, referred to herein as Pei_(wt1)NS₂₃₀, havingamino acid sequence SEQ ID NO:51, assuming an open reading frame inwhich the initiation codon spans from nucleotide 27 through nucleotide29 of SEQ ID NO:50 and the termination codon spans from nucleotide 717through nucleotide 719 of SEQ ID NO:50. The region encodingPei_(wt1)NS₂₃₀, designated nei_(wt1)NS₆₉₀, and having a coding strandcomprising nucleotides 27 to 716 of SEQ ID NO:50 is represented by SEQID NO:52.

PCR amplification of a second nucleic acid molecule encoding a wild typeequine influenza NS protein in the same manner resulted in moleculesnei_(wt2)NS₈₉₁, identical to nei_(wt1)NS₈₉₁ in the coding region; i.e.nei_(wt2)NS₆₉₀, is identical to nei_(wt1)NS₆₉₀. nei_(wt2)NS₈₉₁ differsfrom nei_(wt1)NS₈₉₁ in one nucleotide at base 827 (G to A) which is 111bases downstream from the stop codon. PCR amplification of a thirdnucleic acid encoding a wild type equine influenza NS protein in thesame manner resulted in a nucleic acid molecule of 888 nucleotidesdenoted nei_(wt3)NS₈₈₈, with a coding strand with a nucleic acidsequence designated SEQ ID NO:53. nei_(wt3)NS₈₈₈ is identical tonei_(wt1)NS₈₉₁, except that nei_(wt3)NS₈₈₈, lacks two nucleotides on the5′ end and one nucleotide on the 3′ end. Translation of SEQ ID NO:53indicates that nucleic acid molecule nei_(wt3)NS₈₈₈ also encodesPei_(wt1)NS₂₃₀ (SEQ ID NO:51), assuming an open reading frame having aninitiation codon which spans from nucleotide 25 through nucleotide 27 ofSEQ ID NO:53 and a termination codon which spans from nucleotide 715through nucleotide 717 of SEQ ID NO:53. The nucleic acid molecule havinga coding strand comprising nucleotides 25 to 714 of SEQ ID 53, denotednei_(wt3)NS₆₉₀, is identical to SEQ ID NO:52.

PCR amplification of a fourth nucleic acid of 468 nucleotides encoding aC-terminal portion of the wild type equine influenza NS protein, denotednei_(wt4)NS₄₆₈ and having a coding sequence designated SEQ ID NO:54 wasproduced. Translation of SEQ ID NO:54 indicates that nucleic acidmolecule nei_(wt4)NS₄₆₈ encodes a C-terminal portion of equine influenzaNS protein of about 97 amino acids, referred to herein as Pei_(wt4)NS₉₇,having amino acid sequence SEQ ID NO:55, assuming an open reading framehaving a first codon which spans from nucleotide 3 to 5 of SEQ ID NO:54,and a termination codon spans from nucleotide 294 through 296 of SEQ IDNO:54. Because SEQ ID NO:54 is only a partial gene sequence, it does notcontain an initiation codon. The region encoding Pei_(wt4)NS₉₇,designated nei_(wt4)NS₂₉₃, and having a coding strand comprisingnucleotides 1 to 293 of SEQ ID NO:54, is represented by SEQ ID NO:56.

B. A nucleic acid molecule of 888 nucleotides encoding a cold-adaptedequine influenza virus NS protein, denoted nei_(ca1)NS₈₈₈, with a codingstrand having a sequence designated SEQ ID NO:57 was produced andsequenced as described in Example 16, part A.

Translation of SEQ ID NO:57 indicates that nucleic acid moleculenei_(ca1)NS₈₈₈ encodes a full-length equine influenza NS protein ofabout 230 amino acids, referred to herein as Pei_(ca1)NS₂₃₀, havingamino acid sequence SEQ ID NO:58, assuming an open reading frame inwhich the initiation codon spans from nucleotide 27 through nucleotide29 of SEQ ID NO:57 and the termination codon spans from nucleotide 717through nucleotide 719 of SEQ ID NO:57. The region encodingPei_(ca1)NS₂₃₀, designated nei_(ca1)NS₆₉₀, and having a coding strandcomprising nucleotides 27 to 716 of SEQ ID NO:57, is represented by SEQID NO:59.

PCR amplification of a second nucleic acid molecule encoding acold-adapted equine influenza NS protein in the same manner resulted inmolecules nei_(ca2)NS₈₈₇, containing one less nucleotide at the 3′ endthan nei_(ca1)NS₈₈₈; the coding region nei_(ca2)NS₆₉₀ is identical tonei_(ca1)NS₆₉₀.

C. Comparison of the nucleic acid sequences of the coding strands ofnei_(wt)NS₈₉₁ (SEQ ID NO:50) and nei_(ca1)NS₈₈₈ (SEQ ID NO:57) by DNAalignment reveals the following difference: a A to G shift at base 827which is 111 bases downstream from the stop codon. The 3′ fragmentencoding nei_(wt4)NS₄₆₈ (SEQ ID NO:54) has one shift T to C found atbase 633 relative to the full-length consensus sequence. Comparison ofthe amino acid sequences of proteins Pei_(wt)NS₂₃₀ (SEQ ID NO:51) andPei_(ca1)NS₂₃₀ (SEQ ID NO:58) reveals that there are no differencesbetween amino acid sequences of the wild type and cold-adapted proteins.

EXAMPLE 17

This example describes the cloning and sequencing of equine influenzaPB1 protein (RNA-directed RNA polymerase 1) nucleic acid moleculescorresponding to the N-terminal portion of the protein, for wild type orcold-adapted equine influenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equineinfluenza virus PB1-N proteins were produced as follows. A PCR productcontaining an N-terminal portion of the equine PB1 gene was produced byPCR amplification from equine influenza virus DNA, and primers T7 andREV. A nucleic acid molecule of 1229 nucleotides encoding a wild typePB1-N protein, denoted nei_(wt1)PB1-N₁₂₂₉, with a coding strand having anucleic acid sequence designated SEQ ID NO:62 was produced by furtherPCR amplification using the above described PCR product as a templateand cloned as described in Example 11B. Plasmid DNA was purified andsequenced as in Example 11B, except that only T7 and REV primers wereused in the sequencing kits.

Translation of SEQ ID NO:62 indicates that nucleic acid moleculenei_(wt1)PB1-N₁₂₂₉ encodes an N-terminal portion of influenza PB1protein of about 398 amino acids, referred to herein asPei_(wt1)PB1-N₃₉₈, having amino acid sequence SEQ ID NO:63, assuming anopen reading frame in which the initiation codon spans from nucleotide36 through nucleotide 38 of SEQ ID NO:62, and the last codon spans fromnucleotide 1227 through nucleotide 1229 of SEQ ID NO:62. The regionencoding Pei_(wt1)PB1-N₃₉₈, designated nei_(wt1)PB1-N₁₁₉₄, and having acoding strand comprising nucleotides 36 to 1229 of SEQ ID NO:62 isrepresented by SEQ ID NO:64.

PCR amplification of a second nucleic acid molecule encoding a wild typeequine influenza PB1-N protein in the same manner resulted in a nucleicacid molecule of 673 nucleotides denoted nei_(wt2)PB1-N₆₇₃, with acoding strand with a sequence designated SEQ ID NO:65. Translation ofSEQ ID NO:65 indicates that nucleic acid molecule nei_(wt2)PB1-N₆₇₃encodes Pei_(wt2)PB1-N₂₁₂ (SEQ ID NO:66), assuming an open reading framehaving an initiation codon which spans from nucleotide 36 throughnucleotide 38 of SEQ ID NO:65 and a last codon which spans fromnucleotide 671 through nucleotide 673 of SEQ ID NO:65. Because SEQ IDNO:65 is only a partial gene sequence, it does not contain a stop codon.The nucleic acid molecule having a coding strand comprising nucleotides36 to 671 of SEQ ID NO:65, denoted nei_(wt2)PB1-N₆₃₆, is designated SEQID NO:67.

B. A nucleic acid molecule of 1225 nucleotides encoding an N-terminalportion of influenza PB1 cold-adapted equine influenza virus PB1-Nprotein, denoted nei_(ca1)PB1-N₁₂₂₅, with a coding strand having asequence designated SEQ ID NO:68 was produced, and sequenced asdescribed in as in Example 17, part A.

Translation of SEQ ID NO:68 indicates that nucleic acid moleculenei_(ca1)PB1-N₁₂₂₅ encodes an N-terminal portion of equine influenzaPB-1 protein of about 395 amino acids, referred to herein asPei_(ca1)PB1-N₃₉₅, having amino acid sequence SEQ ID NO:69, assuming anopen reading frame in which the initiation codon spans from nucleotide34 through nucleotide 36 of SEQ ID NO:68, and a last codon which spansfrom nucleotide 1216 through nucleotide 1218 of SEQ ID NO:68. The regionencoding Pei_(ca1)PB1-N₃₉₅, designated nei_(ca1)PB1-N₁₁₈₅, and having acoding strand comprising nucleotides 34 to 1218 of SEQ ID NO:68, isrepresented by SEQ ID NO:70.

PCR amplification of a second nucleic acid molecule encoding acold-adapted equine influenza PB1-N protein in the same manner resultedin molecules nei_(ca2)PB1-N₁₂₂₁, designated SEQ ID NO:71, containingfour less nucleotides at the 5′ end than nei_(ca1)PB1-N₁₂₂₅; the codingregion nei_(ca2)PB1-N₁₁₈₅, is identical to nei_(ca1)PB1-N₁₁₈₅.

C. Comparison of the nucleic acid sequences of the coding strands ofnei_(wt)PB1-N₁₂₂₉ (SEQ ID NO:62) and nei_(ca1)PB1-N₁₂₂₅ (SEQ ID NO:68)by DNA alignment reveals no differences in the coding regions.Comparison of the amino acid sequences of proteins Pei_(wt)PB1-N₃₉₅ (SEQID NO:63) and Pei_(ca1)PB1-N₃₉₅ (SEQ ID NO:69) also reveals nodifferences.

EXAMPLE 18

This example describes the cloning and sequencing of equine influenzaPB1 protein (RNA-directed RNA polymerasel) nucleic acid moleculescorresponding to the C-terminal portion of the protein, for wild type orcold-adapted equine influenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equineinfluenza virus PB1-C proteins were produced as follows. A PCR productcontaining an C-terminal portion of the equine PB1 gene was produced byPCR amplification from equine influenza virus DNA, and primer w569designated SEQ ID NO:102. A nucleic acid molecule of 1234 nucleotidesencoding a wild type PB1-C protein, denoted nei_(wt1)PB1-C₁₂₃₄, with acoding strand having a nucleic acid sequence designated SEQ ID NO:85 wasproduced by further PCR amplification using the above described PCRproduct as a template and cloned as described in Example 11B. PlasmidDNA was purified and sequenced as in Example 11A, except that differentprimers were used in the sequencing kits. T7, REV, w569, efPB1-a1,designated SEQ ID NO:97, efPB1-a2, designated SEQ ID NO:98, efPB1-s1,designated SEQ ID NO:99, efPB1-s2, designated SEQ ID NO:100, andefPB1-s3, designated SEQ ID NO:101 were used in one instance, T7, REV,efPB1-a1, efPB1-a2,efPB1-s1, efPB1-s2, and efPB1-s3 were used in anotherinstance and T7 and REV were used in another instance.

Translation of SEQ ID NO:85 indicates that nucleic acid moleculenei_(wt1)PB1-C₁₂₃₄ encodes an C-terminal portion of influenza PB1protein of about 396 amino acids, referred to herein asPei_(wt1)PB1-C₃₉₆, having amino acid sequence SEQ ID NO:86, assuming anopen reading frame in which the first codon spans from nucleotide 1through nucleotide 3 of SEQ ID NO:85 and a termination codon which spansfrom nucleotide 1189 through nucleotide 1191 of SEQ ID NO:85. BecauseSEQ ID NO:85 is only a partial gene sequence, it does not contain aninitiation codon. The region encoding Pei_(wt1)PB1-C₃₉₆, designatednei_(wt1)PB1-C₁₁₈₈, and having a coding strand comprising nucleotides 1to 1188 of SEQ ID NO:85 is represented by SEQ ID NO:87.

PCR amplification of a second nucleic acid molecule encoding a wild typeequine influenza PB1-C protein in the same manner resulted in a nucleicacid molecule of 1240 nucleotides denoted nei_(wt2)PB1-C₁₂₄₀, with acoding strand with a sequence designated SEQ ID NO:88. Translation ofSEQ ID NO:88 indicates that nucleic acid molecule nei_(wt2)PB1-N₁₂₄₀encodes a molecule designated Pei_(wt2)PB1-C₃₉₆ (SEQ ID NO:89) whichdiffers from Pei_(wt1)PB1-C₃₉₆(SEQ ID NO:85) in one nucleotide.Nucleotide 382 of nei_(wt1)PB1-C₁₂₃₄, i.e. nucleotide 382 ofnei_(wt1)PB1-C₁₁₈₈ was A, while nucleotide 389 of nei_(wt2)PB1-C₁₂₄₀,i.e. nucleotide 382 of nei_(wt2)PB1-C₁₁₈₈ was T. Translation ofnei_(wt2)PB1-C₁₂₄₀ results in an amino acid change of T to S.

B. A nucleic acid molecule of 1241 nucleotides encoding an C-terminalportion of influenza PB1 cold-adapted equine influenza virus PB1-Cprotein, denoted nei_(ca1)PB1-C₁₂₄₁, with a coding strand having asequence designated SEQ ID NO:91 was produced, and sequenced asdescribed in as in Example 18, part A.

Translation of SEQ ID NO:91 indicates that nucleic acid moleculenei_(ca1)PB1-C₁₂₄₁ encodes an C-terminal portion of equine influenzaPB-1 protein of about 396 amino acids, referred to herein asPei_(ca1)PB1-C₃₉₆, having amino acid sequence SEQ ID NO:92, assuming anopen reading frame in which the first codon spans from nucleotide 8through nucleotide 10 of SEQ ID NO:91 and a termination codon that spansfrom nucleotide 1196 through nucleotide 1198 of SEQ ID NO:91. BecauseSEQ ID NO:91 is only a partial gene sequence, it does not contain aninitiation codon. The region encoding Pei_(ca1)PB1-C₃₉₆, designatednei_(ca1)PB1-C₁₁₈₈, and having a coding strand comprising nucleotides 8to 1195 of SEQ ID NO:91, is represented by SEQ ID NO:93.

PCR amplification of a second nucleic acid molecule encoding acold-adapted equine influenza PB1-C protein in the same manner resultedin a nucleic acid molecule of 1241 nucleotides denotednei_(ca2)PB1-C₁₂₄₁, with a coding strand with a sequence designated SEQID NO:94. Translation of SEQ ID NO:94 indicates that nucleic acidmolecule nei_(ca2)PB1-C₁₂₄₁ encodes a molecule designatedPei_(ca2)PB1-C₃₉₆ (SEQ ID NO:95) which differs from Pei_(ca1)PB1-C₃₉₆(SEQ ID NO:92) in one nucleotide. Nucleotide 1044 of nei_(ca1)PB1-C₁₂₄₁,i.e. nucleotide 1037 of nei_(ca1)PB1-N₁₁₈₈ was A, while nucleotide 1044of nei_(ca2)PB1-C₁₂₄₁, i.e. nucleotide 1037 of nei_(ca2)PB1-C₁₁₈₈ was G.Translation of nei_(ca2)PB1-C₁₂₄₁ results in an amino acid change of Rto K.

C. Comparison of the nucleic acid sequences of the coding strands ofnei_(wt1)PB1-C₁₂₃₄ (SEQ ID NO:85) and nei_(ca1)PB1-C₁₂₄₁ (SEQ ID NO:91)by DNA alignment reveals the following differences: a C to T shift atbase 600 of SEQ ID NO:85, and a T to A shift at base 603 of SEQ IDNO:85. Comparison of the amino acid sequences of proteinsPei_(wt1)PB1-C₃₉₆ (SEQ ID NO:86) and Pei_(ca1)PB1-N₃₉₆ (SEQ ID NO:92)reveals the following difference: a H to Q amino acid shift 203 whenrelating to the T to A base shift at base 603 in the DNA sequences.There is no amino acid shift resulting from the C to T base shift atbase 600.

EXAMPLE 19

This example describes the cloning and sequencing of equine influenzaPB1 protein (RNA-directed RNA polymerase) nucleic acid molecules forwild type or cold-adapted equine influenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equineinfluenza virus PB1 proteins were produced as follows. The wild type orcold-adapted equine influenza genes were cloned in two fragments, theN-terminal portion was produced as in Example 17 and the C-terminalportion of the gene was produced as in Example 18. The DNA sequence forthe wild type equine influenza PB1 gene was generated by combining thesequences for the wild type PB1-N protein, nei_(wt1)PB1-N₁₂₂₉ (SEQ IDNO:62) and nei_(wt2)PB1-N₆₇₃ (SEQ ID NO:65) with the gene fragments forthe wild type PB1-C protein, denoted nei_(wt1)PB1-C₁₂₃₄ (SEQ ID NO:85)and nei_(wt2)PB1-C₁₂₄₀ (SEQ ID NO:88). The result of combining theN-terminal and C-terminal portions of the PB1 wild type influenza virusyielded a complete DNA sequence of 2341 nucleotides denotednei_(wt)PB1₂₃₄₁(SEQ ID NO:103). Translation of SEQ ID NO:103 indicatesthat the nucleic acid molecule nei_(wt)PB2₂₃₄₁ encodes a full lengthequine influenza PB1 protein of about 757 amino acids referred to hereinas Pei_(wt)PB1₇₅₇, having amino acid sequence SEQ ID NO:104, assuming anopen reading frame in which the initiation codon spans from nucleotide25 through nucleotide 27of SEQ ID NO:103 and the termination codon spansfrom nucleotide 2293 through nucleotide 2295 of SEQ ID NO:103. Theregion encoding Pei_(wt)PB1₇₅₇ designated nei_(wt)PB1₂₂₇₁, and having acoding strand comprising nucleotides 25 to 2292 of SEQ ID NO: 103, isSEQ ID NO:105.

B. A DNA sequence of 2341 nucleotides encoding a cold-adapted equineinfluenza virus PB1, denoted nei_(ca1)PB1₂₃₄₁, with a sequence denotedSEQ ID NO:106 was produced by combining the sequences for the N-terminaland C-terminal portions of the PB1 cold-adapted equine influenza gene.The clones for the N-terminal sequences are denoted nei_(ca1)PB1-N₁₂₂₅(SEQ ID NO:68) and nei_(ca2)PB1-N₁₂₂₁ (SEQ ID NO:71). The clones for theC-terminal sequences are denoted nei_(ca1)PB1-C₁₂₄₁ (SEQ ID NO:91) andnei_(ca2)PB1-C₁₂₄₁, (SEQ ID NO:94).

Translation of SEQ ID NO:106 indicates that nucleic acid moleculenei_(ca1)PB1₂₃₄₁ encodes a full-length equine influenza PB1 protein ofabout 757 amino acids, referred to herein as Pei_(ca1)PB1₇₅₇, havingamino acid sequence SEQ ID NO:107, assuming an open reading frame inwhich the initiation codon spans from nucleotide 25 through nucleotide27 SEQ ID NO:106 and the termination codon spans from nucleotide 2296through nucleotide 2298 of SEQ ID NO:106. The region encodingPei_(ca1)PB1₇₅₇ designated nei_(ca1)PB1₂₂₇₁ and having a coding strandcomprising nucleotides 25 to 2295 of SEQ ID NO:108.

C. Comparison of the nucleic acid sequences of the coding strands ofnei_(wt)PB1₂₃₄₁ (SEQ ID NO:103) and nei_(ca1)PB1₂₃₄₁ (SEQ ID NO:106) byDNA alignment reveals the following differences: a C to T base shift atbase 1683, and a T to A base shift at base 1686. Comparison of the aminoacid sequences of proteins Pei_(wt)PB1₇₅₇ (SEQ ID NO:104) andPei_(ca1)PB1₇₅₇ (SEQ ID NO:107) reveals the following differences: noshift in base C at amino acid 561 relating to the C to T shift at base1683 , and a H to Q shift at amino acid 562 relating to the a T to Ashift at base 1683 in the DNA sequence.

EXAMPLE 20

This example describes the cloning and sequencing of equine influenza PAprotein (RNA polymerase A) nucleic acid molecules corresponding to theC-terminal portion of the protein, for wild type or cold-adapted equineinfluenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equineinfluenza virus PA-C proteins were produced as follows. A PCR productcontaining the C-terminal portion of the equine PA gene was produced byPCR amplification using from equine influenza virus DNA and primers C+PAand C−PA, designated SEQ ID NO:83 and SEQ ID NO:84 respectively. Anucleic acid molecule of 1228 nucleotides encoding a wild type PA-Cprotein, denoted nei_(wt1)PA-C₁₂₂₈, with a coding strand having anucleic acid sequence designated SEQ ID NO:76 was produced by furtherPCR amplification using the above-described PCR product as a templateand cloned as described in Example 11B. Plasmid DNA was purified andsequenced as in Example 11A, except that different primers were used inthe sequencing kits. T7 and REV were used in one instance; PAC-1,designated SEQ ID NO:72, PAC-2, designated SEQ ID NO:73, PAC-3,designated SEQ ID NO:74, PAC-4, designated SEQ ID NO:75, T7 and REV wereused in another instance; and PAC-1, PAC-2, T7 and REV were used inanother instance.

Translation of SEQ ID NO:76 indicates that nucleic acid moleculenei_(wt1)PA-C₁₂₂₈ encodes a C-terminal portion of influenza PA proteinof about 388 amino acids, referred to herein as Pei_(wt1)PA-C₃₈₈, havingamino acid sequence SEQ ID NO:77, assuming an open reading frame havinga first codon spans from nucleotide 3 through nucleotide 5 of SEQ IDNO:76 and a termination codon which spans from nucleotide 1167 throughnucleotide 1169 of SEQ ID NO:76. Because SEQ ID NO:76 is only a partialgene sequence, it does not contain an initiation codon. The regionencoding Pei_(wt1)PA-C₃₈₈, designated nei_(wt1)PA-C₁₁₆₄, and having acoding strand comprising nucleotides 3 to 1166 of SEQ ID NO:76 isrepresented by SEQ ID NO:78.

PCR amplification of a second nucleic acid molecule encoding a wild typeequine influenza PA-C protein in the same manner resulted in a nucleicacid molecule of 1223 nucleotides denoted nei_(wt2)PA-C₁₂₂₃, with acoding strand with a sequence designated SEQ ID NO:79. nei_(wt2)PA-C₁₂₂₃is identical to nei_(wt1)PA-C₁₂₂₈, with the exception of a T to C baseshift at base 753 and that nei_(wt2)PA-C₁₂₂₃ lacks five nucleotides onthe 3′-end. Translation of SEQ ID NO:79 indicates that nucleic acidmolecule nei_(wt2)PA-C₁₂₂₃ also encodes Pei_(wt1)PA-C₃₈₈ (SEQ ID NO:77),assuming an open reading frame having a first codon which spans fromnucleotide 3 through nucleotide 5 of SEQ ID NO:79 and a terminationcodon which spans from nucleotide 1167 through nucleotide 1169 of SEQ IDNO:79. Because SEQ ID NO:79 is only a partial gene sequence, it does notcontain an initiation codon. The nucleic acid molecule having a codingstrand comprising nucleotides 3 to 1166 of SEQ ID NO:79, denotednei_(wt2)PA-C₁₂₂₃, is identical to SEQ ID NO 78.

B. A nucleic acid molecule of 1233 nucleotides encoding a C-terminalportion of influenza PA-C cold-adapted equine influenza virus protein,denoted nei_(ca1)PA-C₁₂₃₃, and having a coding strand having a sequencedesignated SEQ ID NO:80 was produced as described in as in Example 20,part A, except that the pCR®-Blunt cloning vector was used.

Translation of SEQ ID NO:80 indicates that nucleic acid moleculenei_(ca1)PA-C₁₂₃₃ encodes a C-terminal portion of equine influenza PAprotein of about 390 amino acids, referred to herein asPei_(ca1)PA-C₃₉₀, having amino acid sequence SEQ ID NO:81, assuming anopen reading frame having a first codon which spans from nucleotide 3through nucleotide 5 of SEQ ID NO:80 and a termination codon which spansfrom nucleotide 1173 through nucleotide 1175 of SEQ ID NO:80. BecauseSEQ ID NO:80 is only a partial gene sequence, it does not contain aninitiation codon. The region encoding Pei_(ca1)PA-C₃₉₀, designatednei_(ca1)PA-C₁₁₇₀, and having a coding strand comprising nucleotides 3to 1172 of SEQ ID NO:80, is represented by SEQ ID NO:82.

PCR amplification of a second nucleic acid molecule encoding acold-adapted equine influenza PA-C protein in the same manner resultedin molecule nei_(ca2)PA-C₁₂₃₃, containing one A to G base shift at base953 as compared to nei_(ca1)PA-C₁₂₃₃; this base shift does not result inan amino acid change so Pei_(ca2)PA-C₃₉₀, is identical toPei_(ca1)PA-C₃₉₀ (SEQ ID NO:81.)

C. Comparison of the nucleic acid sequences of the coding strands ofnei_(wt1)PA-C₁₂₂₈ (SEQ ID NO:76) and nei_(ca1)PA-C₁₂₃₃ (SEQ ID NO:80) byDNA alignment reveals the following difference: an C to T base shift atbase 753 of SEQ ID NO:80. Comparison of the amino acid sequences ofproteins Pei_(wt1)PA-C₃₈₈ (SEQ ID NO:77) and Pei_(ca1)PA-₃₉₀ (SEQ IDNO:81) reveals the following difference: a W to R shift at amino acid251 when relating to the C to T base shift at base 753 in the DNAsequences.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims.

108 1 1023 DNA Equine influenza virus H3N8 CDS (25)..(780) At location663, r = A or G; At amino acid location 213, Xaa = Val 1 gcaaaagcaggtagatattt aaag atg agt ctt ctg acc gag gtc gaa acg 51 Met Ser Leu LeuThr Glu Val Glu Thr 1 5 tac gtt ctc tct atc gta cca tca ggc ccc ctc aaagcc gag atc gcg 99 Tyr Val Leu Ser Ile Val Pro Ser Gly Pro Leu Lys AlaGlu Ile Ala 10 15 20 25 cag aga ctt gaa gat gtc ttt gca ggg aag aac accgat ctt gag gca 147 Gln Arg Leu Glu Asp Val Phe Ala Gly Lys Asn Thr AspLeu Glu Ala 30 35 40 ctc atg gaa tgg cta aag aca aga cca atc ctg tca cctctg act aaa 195 Leu Met Glu Trp Leu Lys Thr Arg Pro Ile Leu Ser Pro LeuThr Lys 45 50 55 ggg att tta gga ttc gta ttc acg ctc acc gtg ccc agt gagcga gga 243 Gly Ile Leu Gly Phe Val Phe Thr Leu Thr Val Pro Ser Glu ArgGly 60 65 70 ctg cag cgt aga cgc ttt gtc caa aat gcc ctt agt gga aac ggagat 291 Leu Gln Arg Arg Arg Phe Val Gln Asn Ala Leu Ser Gly Asn Gly Asp75 80 85 cca aac aac atg gac aga gca gta aaa ctg tac agg aag ctt aaa aga339 Pro Asn Asn Met Asp Arg Ala Val Lys Leu Tyr Arg Lys Leu Lys Arg 9095 100 105 gaa ata aca ttc cat ggg gca aaa gag gtg gca ctc agc tat tccact 387 Glu Ile Thr Phe His Gly Ala Lys Glu Val Ala Leu Ser Tyr Ser Thr110 115 120 ggt gca cta gcc agc tgc atg gga ctc ata tac aac aga atg ggaact 435 Gly Ala Leu Ala Ser Cys Met Gly Leu Ile Tyr Asn Arg Met Gly Thr125 130 135 gtg aca acc gaa gtg gca ttt ggc ctg gta tgc gcc aca tgt gaacag 483 Val Thr Thr Glu Val Ala Phe Gly Leu Val Cys Ala Thr Cys Glu Gln140 145 150 atc gct gat tcc cag cat cga tct cac agg cag atg gtg aca acaacc 531 Ile Ala Asp Ser Gln His Arg Ser His Arg Gln Met Val Thr Thr Thr155 160 165 aac cca tta atc aga cat gaa aac aga atg gta tta gcc agt accacg 579 Asn Pro Leu Ile Arg His Glu Asn Arg Met Val Leu Ala Ser Thr Thr170 175 180 185 gct aaa gcc atg gag cag atg gca ggg tcg agt gag cag gcagca gag 627 Ala Lys Ala Met Glu Gln Met Ala Gly Ser Ser Glu Gln Ala AlaGlu 190 195 200 gcc atg gag gtt gct agt aag gct agg cag atg gtr cag gcaatg aga 675 Ala Met Glu Val Ala Ser Lys Ala Arg Gln Met Xaa Gln Ala MetArg 205 210 215 acc att ggg acc cac cct agc tcc agt gcc ggt ttg aaa gatgat ctc 723 Thr Ile Gly Thr His Pro Ser Ser Ser Ala Gly Leu Lys Asp AspLeu 220 225 230 ctt gaa aat ttg cag gcc tac cag aaa cgg atg gga gtg caaatg cag 771 Leu Glu Asn Leu Gln Ala Tyr Gln Lys Arg Met Gly Val Gln MetGln 235 240 245 cga ttc aag tgatcctctc gttattgcag caagtatcat tgggatcttg820 Arg Phe Lys 250 cacttgatat tgtggattct tgatcgcctt ttcttcaaattcatttatcg tcgccttaaa 880 tacgggttga aaagagggcc ttctacggaa ggagtacctgagtctatgag ggaagaatat 940 cggcaggaac agcagaatgc tgtggatgtt gacgatggtcattttgtcaa catagagctg 1000 gagtaaaaaa ctaccttgtt tct 1023 2 252 PRTEquine influenza virus H3N8 At amino acid location 213, Xaa = Val 2 MetSer Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Val Pro 1 5 10 15Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe 20 25 30Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr 35 40 45Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe 50 55 60Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val 65 70 7580 Gln Asn Ala Leu Ser Gly Asn Gly Asp Pro Asn Asn Met Asp Arg Ala 85 9095 Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu Ile Thr Phe His Gly Ala 100105 110 Lys Glu Val Ala Leu Ser Tyr Ser Thr Gly Ala Leu Ala Ser Cys Met115 120 125 Gly Leu Ile Tyr Asn Arg Met Gly Thr Val Thr Thr Glu Val AlaPhe 130 135 140 Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser GlnHis Arg 145 150 155 160 Ser His Arg Gln Met Val Thr Thr Thr Asn Pro LeuIle Arg His Glu 165 170 175 Asn Arg Met Val Leu Ala Ser Thr Thr Ala LysAla Met Glu Gln Met 180 185 190 Ala Gly Ser Ser Glu Gln Ala Ala Glu AlaMet Glu Val Ala Ser Lys 195 200 205 Ala Arg Gln Met Xaa Gln Ala Met ArgThr Ile Gly Thr His Pro Ser 210 215 220 Ser Ser Ala Gly Leu Lys Asp AspLeu Leu Glu Asn Leu Gln Ala Tyr 225 230 235 240 Gln Lys Arg Met Gly ValGln Met Gln Arg Phe Lys 245 250 3 756 DNA Equine influenza virus H3N8 3atgagtcttc tgaccgaggt cgaaacgtac gttctctcta tcgtaccatc aggccccctc 60aaagccgaga tcgcgcagag acttgaagat gtctttgcag ggaagaacac cgatcttgag 120gcactcatgg aatggctaaa gacaagacca atcctgtcac ctctgactaa agggatttta 180ggattcgtat tcacgctcac cgtgcccagt gagcgaggac tgcagcgtag acgctttgtc 240caaaatgccc ttagtggaaa cggagatcca aacaacatgg acagagcagt aaaactgtac 300aggaagctta aaagagaaat aacattccat ggggcaaaag aggtggcact cagctattcc 360actggtgcac tagccagctg catgggactc atatacaaca gaatgggaac tgtgacaacc 420gaagtggcat ttggcctggt atgcgccaca tgtgaacaga tcgctgattc ccagcatcga 480tctcacaggc agatggtgac aacaaccaac ccattaatca gacatgaaaa cagaatggta 540ttagccagta ccacggctaa agccatggag cagatggcag ggtcgagtga gcaggcagca 600gaggccatgg aggttgctag taaggctagg cagatggtrc aggcaatgag aaccattggg 660acccacccta gctccagtgc cggtttgaaa gatgatctcc ttgaaaattt gcaggcctac 720cagaaacgga tgggagtgca aatgcagcga ttcaag 756 4 1023 DNA Equine influenzavirus H3N8 CDS (25)..(780) 4 gcaaaagcag gtagatattt aaag atg agt ctt ctgacc gag gtc gaa acg 51 Met Ser Leu Leu Thr Glu Val Glu Thr 1 5 tac gttctc tct atc tta cca tca ggc ccc ctc aaa gcc gag atc gcg 99 Tyr Val LeuSer Ile Leu Pro Ser Gly Pro Leu Lys Ala Glu Ile Ala 10 15 20 25 cag agactt gaa gat gtc ttt gca ggg aag aac acc gat ctt gag gca 147 Gln Arg LeuGlu Asp Val Phe Ala Gly Lys Asn Thr Asp Leu Glu Ala 30 35 40 ctc atg gaatgg cta aag aca aga cca atc ctg tca cct ctg act aaa 195 Leu Met Glu TrpLeu Lys Thr Arg Pro Ile Leu Ser Pro Leu Thr Lys 45 50 55 ggg att tta ggattc gta ttc acg ctc acc gtg ccc agt gag cga gga 243 Gly Ile Leu Gly PheVal Phe Thr Leu Thr Val Pro Ser Glu Arg Gly 60 65 70 ctg cag cgt aga cgcttt gtc caa aat gcc ctt agt gga aac gga gat 291 Leu Gln Arg Arg Arg PheVal Gln Asn Ala Leu Ser Gly Asn Gly Asp 75 80 85 cca aac aac atg gac agagca gta aaa ctg tac agg aag ctt aaa aga 339 Pro Asn Asn Met Asp Arg AlaVal Lys Leu Tyr Arg Lys Leu Lys Arg 90 95 100 105 gaa ata aca ttc catggg gca aaa gag gtg gca ctc agc tat tcc act 387 Glu Ile Thr Phe His GlyAla Lys Glu Val Ala Leu Ser Tyr Ser Thr 110 115 120 ggt gca cta gcc agctgc atg gga ctc ata tac aac aga atg gga act 435 Gly Ala Leu Ala Ser CysMet Gly Leu Ile Tyr Asn Arg Met Gly Thr 125 130 135 gtg aca acc gaa gtggca ttt ggc ctg gta tgc gcc aca tgt gaa cag 483 Val Thr Thr Glu Val AlaPhe Gly Leu Val Cys Ala Thr Cys Glu Gln 140 145 150 atc gct gat tcc cagcat cga tct cac agg cag atg gtg aca ata acc 531 Ile Ala Asp Ser Gln HisArg Ser His Arg Gln Met Val Thr Ile Thr 155 160 165 aac cca tta atc agacat gaa aac aga atg gta tta gcc agt acc acg 579 Asn Pro Leu Ile Arg HisGlu Asn Arg Met Val Leu Ala Ser Thr Thr 170 175 180 185 gct aaa gcc atggag cag atg gca ggg tcg agt gag cag gca gca gag 627 Ala Lys Ala Met GluGln Met Ala Gly Ser Ser Glu Gln Ala Ala Glu 190 195 200 gcc atg gag gttgct agt aag gct agg cag atg gta cag gca atg aga 675 Ala Met Glu Val AlaSer Lys Ala Arg Gln Met Val Gln Ala Met Arg 205 210 215 acc att ggg acccac cct agc tcc agt gcc ggt ttg aaa gat gat ctc 723 Thr Ile Gly Thr HisPro Ser Ser Ser Ala Gly Leu Lys Asp Asp Leu 220 225 230 ctt gaa aat ttgcag gcc tac cag aaa cgg atg gga gtg caa atg cag 771 Leu Glu Asn Leu GlnAla Tyr Gln Lys Arg Met Gly Val Gln Met Gln 235 240 245 cga ttc aagtgatcctctc gttattgcag caagtatcat tgggatcttg 820 Arg Phe Lys 250cacttgatat tgtggattct tgatcgcctt ttcttcaaat tcatttatcg tcgccttaaa 880tacggcttga aaagagggcc ttctacggaa ggagtacctg agtctatgag ggaagaatat 940cggcaggaac agcagaatgc tgtggatgtt gacgatggtc attttgtcaa catagagctg 1000gagtaaaaaa ctaccttgtt tct 1023 5 252 PRT Equine influenza virus H3N8 5Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Leu Pro 1 5 1015 Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe 20 2530 Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr 35 4045 Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe 50 5560 Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val 65 7075 80 Gln Asn Ala Leu Ser Gly Asn Gly Asp Pro Asn Asn Met Asp Arg Ala 8590 95 Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu Ile Thr Phe His Gly Ala100 105 110 Lys Glu Val Ala Leu Ser Tyr Ser Thr Gly Ala Leu Ala Ser CysMet 115 120 125 Gly Leu Ile Tyr Asn Arg Met Gly Thr Val Thr Thr Glu ValAla Phe 130 135 140 Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp SerGln His Arg 145 150 155 160 Ser His Arg Gln Met Val Thr Ile Thr Asn ProLeu Ile Arg His Glu 165 170 175 Asn Arg Met Val Leu Ala Ser Thr Thr AlaLys Ala Met Glu Gln Met 180 185 190 Ala Gly Ser Ser Glu Gln Ala Ala GluAla Met Glu Val Ala Ser Lys 195 200 205 Ala Arg Gln Met Val Gln Ala MetArg Thr Ile Gly Thr His Pro Ser 210 215 220 Ser Ser Ala Gly Leu Lys AspAsp Leu Leu Glu Asn Leu Gln Ala Tyr 225 230 235 240 Gln Lys Arg Met GlyVal Gln Met Gln Arg Phe Lys 245 250 6 756 DNA Equine influenza virusH3N8 6 atgagtcttc tgaccgaggt cgaaacgtac gttctctcta tcttaccatc aggccccctc60 aaagccgaga tcgcgcagag acttgaagat gtctttgcag ggaagaacac cgatcttgag 120gcactcatgg aatggctaaa gacaagacca atcctgtcac ctctgactaa agggatttta 180ggattcgtat tcacgctcac cgtgcccagt gagcgaggac tgcagcgtag acgctttgtc 240caaaatgccc ttagtggaaa cggagatcca aacaacatgg acagagcagt aaaactgtac 300aggaagctta aaagagaaat aacattccat ggggcaaaag aggtggcact cagctattcc 360actggtgcac tagccagctg catgggactc atatacaaca gaatgggaac tgtgacaacc 420gaagtggcat ttggcctggt atgcgccaca tgtgaacaga tcgctgattc ccagcatcga 480tctcacaggc agatggtgac aataaccaac ccattaatca gacatgaaaa cagaatggta 540ttagccagta ccacggctaa agccatggag cagatggcag ggtcgagtga gcaggcagca 600gaggccatgg aggttgctag taaggctagg cagatggtac aggcaatgag aaccattggg 660acccacccta gctccagtgc cggtttgaaa gatgatctcc ttgaaaattt gcaggcctac 720cagaaacgga tgggagtgca aatgcagcga ttcaag 756 7 1762 DNA Equine influenzavirus H3N8 CDS (30)..(1724) 7 agcaaaagca ggggatattt ctgtcaatc atg aagaca acc att att ttg ata 53 Met Lys Thr Thr Ile Ile Leu Ile 1 5 cca ctgacc cat tgg gtc tac agt caa aac cca acc agt ggc aac aac 101 Pro Leu ThrHis Trp Val Tyr Ser Gln Asn Pro Thr Ser Gly Asn Asn 10 15 20 aca gcc acatta tgt ctg gga cac cat gca gta gca aat gga aca ttg 149 Thr Ala Thr LeuCys Leu Gly His His Ala Val Ala Asn Gly Thr Leu 25 30 35 40 gta aaa acaata act gat gac caa att gag gtg aca aat gct act gaa 197 Val Lys Thr IleThr Asp Asp Gln Ile Glu Val Thr Asn Ala Thr Glu 45 50 55 tta gtt cag agcatt tca ata ggg aaa ata tgc aac aac tca tat aga 245 Leu Val Gln Ser IleSer Ile Gly Lys Ile Cys Asn Asn Ser Tyr Arg 60 65 70 gtt cta gat gga agaaat tgc aca tta ata gat gca atg cta gga gac 293 Val Leu Asp Gly Arg AsnCys Thr Leu Ile Asp Ala Met Leu Gly Asp 75 80 85 ccc cac tgt gat gtc tttcag tat gag aat tgg gac ctc ttc ata gaa 341 Pro His Cys Asp Val Phe GlnTyr Glu Asn Trp Asp Leu Phe Ile Glu 90 95 100 aga agc agc gct ttc agcagt tgc tac cca tat gac atc cct gac tat 389 Arg Ser Ser Ala Phe Ser SerCys Tyr Pro Tyr Asp Ile Pro Asp Tyr 105 110 115 120 gca tcg ctc cgg tccatt gta gca tcc tca gga aca ttg gaa ttc aca 437 Ala Ser Leu Arg Ser IleVal Ala Ser Ser Gly Thr Leu Glu Phe Thr 125 130 135 gca gag gga ttc acatgg aca ggt gtc act caa aac gga aga agt gga 485 Ala Glu Gly Phe Thr TrpThr Gly Val Thr Gln Asn Gly Arg Ser Gly 140 145 150 tcc tgc aaa agg ggatca gcc gat agt ttc ttt agc cga ctg aat tgg 533 Ser Cys Lys Arg Gly SerAla Asp Ser Phe Phe Ser Arg Leu Asn Trp 155 160 165 cta aca gaa tct ggaaac tct tac ccc aca ttg aat gtg aca atg cct 581 Leu Thr Glu Ser Gly AsnSer Tyr Pro Thr Leu Asn Val Thr Met Pro 170 175 180 aac aat aaa aat ttcgac aaa cta tac atc tgg ggg att cat cac ccg 629 Asn Asn Lys Asn Phe AspLys Leu Tyr Ile Trp Gly Ile His His Pro 185 190 195 200 agc tca aac aaagag cag aca aaa ttg tac atc caa gaa tcg gga cga 677 Ser Ser Asn Lys GluGln Thr Lys Leu Tyr Ile Gln Glu Ser Gly Arg 205 210 215 gta aca gtc tcaaca aaa aga agt caa caa aca ata atc cct aac atc 725 Val Thr Val Ser ThrLys Arg Ser Gln Gln Thr Ile Ile Pro Asn Ile 220 225 230 gga tct aga ccgcgg gtc agg ggt caa tca ggc agg ata agc ata tac 773 Gly Ser Arg Pro ArgVal Arg Gly Gln Ser Gly Arg Ile Ser Ile Tyr 235 240 245 tgg acc att gtaaaa cct gga gat atc cta atg ata aac agt aat ggc 821 Trp Thr Ile Val LysPro Gly Asp Ile Leu Met Ile Asn Ser Asn Gly 250 255 260 aac tta gtt gcaccg cgg gga tat ttt aaa ttg aaa aca ggg aaa agc 869 Asn Leu Val Ala ProArg Gly Tyr Phe Lys Leu Lys Thr Gly Lys Ser 265 270 275 280 tct gta atgaga tca gat gca ccc ata gac att tgt gtg tct gaa tgt 917 Ser Val Met ArgSer Asp Ala Pro Ile Asp Ile Cys Val Ser Glu Cys 285 290 295 att aca ccaaat gga agc atc ccc aac gac aaa cca ttt caa aat gtg 965 Ile Thr Pro AsnGly Ser Ile Pro Asn Asp Lys Pro Phe Gln Asn Val 300 305 310 aac aaa gttaca tat gga aaa tgc ccc aag tat atc agg caa aac act 1013 Asn Lys Val ThrTyr Gly Lys Cys Pro Lys Tyr Ile Arg Gln Asn Thr 315 320 325 tta aag ctggcc act ggg atg agg aat gta cca gaa aag caa atc aga 1061 Leu Lys Leu AlaThr Gly Met Arg Asn Val Pro Glu Lys Gln Ile Arg 330 335 340 gga atc tttgga gca ata gcg gga ttc ata gaa aac ggc tgg gaa gga 1109 Gly Ile Phe GlyAla Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly 345 350 355 360 atg gttgat ggg tgg tat gga ttc cga tat caa aac tcg gaa gga aca 1157 Met Val AspGly Trp Tyr Gly Phe Arg Tyr Gln Asn Ser Glu Gly Thr 365 370 375 gga caagct gca gat cta aag agc act caa gca gcc atc gac cag atc 1205 Gly Gln AlaAla Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile 380 385 390 aat ggaaaa tta aac aga gtg att gaa agg acc aat gag aaa ttc cat 1253 Asn Gly LysLeu Asn Arg Val Ile Glu Arg Thr Asn Glu Lys Phe His 395 400 405 caa atagag aag gaa ttc tca gaa gta gaa ggg agg atc cag gac ttg 1301 Gln Ile GluLys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu 410 415 420 gag aagtat gta gaa gac acc aaa ata gac cta tgg tcc tac aat gca 1349 Glu Lys TyrVal Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala 425 430 435 440 gaattg ctg gtg gct cta aaa aat caa cat aca att gac tta aca gat 1397 Glu LeuLeu Val Ala Leu Lys Asn Gln His Thr Ile Asp Leu Thr Asp 445 450 455 gcagaa atg aat aaa tta ttc gag aag act aga cgc cag tta aga gaa 1445 Ala GluMet Asn Lys Leu Phe Glu Lys Thr Arg Arg Gln Leu Arg Glu 460 465 470 aacgcg gaa gac atg gga ggt gga tgt ttc aag ata tac cac aaa tgt 1493 Asn AlaGlu Asp Met Gly Gly Gly Cys Phe Lys Ile Tyr His Lys Cys 475 480 485 gataat gca tgc att gga tca ata aga aat ggg aca tat gac cat tac 1541 Asp AsnAla Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Tyr 490 495 500 atatac aga gat gaa gca tta aac aac cgg ttt caa atc aaa ggt gtt 1589 Ile TyrArg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val 505 510 515 520gag ttg aaa tca ggc tac aaa gat tgg ata ctg tgg att tca ttc gcc 1637 GluLeu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala 525 530 535ata tca tgc ttc tta att tgc gtt gtt cta ttg ggt ttc att atg tgg 1685 IleSer Cys Phe Leu Ile Cys Val Val Leu Leu Gly Phe Ile Met Trp 540 545 550gct tgc caa aaa ggc aac atc aga tgc aac att tgc att tgagtaaact 1734 AlaCys Gln Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile 555 560 565 gatagttaaaaacacccttg tttctact 1762 8 565 PRT Equine influenza virus H3N8 8 Met LysThr Thr Ile Ile Leu Ile Pro Leu Thr His Trp Val Tyr Ser 1 5 10 15 GlnAsn Pro Thr Ser Gly Asn Asn Thr Ala Thr Leu Cys Leu Gly His 20 25 30 HisAla Val Ala Asn Gly Thr Leu Val Lys Thr Ile Thr Asp Asp Gln 35 40 45 IleGlu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ile Ser Ile Gly 50 55 60 LysIle Cys Asn Asn Ser Tyr Arg Val Leu Asp Gly Arg Asn Cys Thr 65 70 75 80Leu Ile Asp Ala Met Leu Gly Asp Pro His Cys Asp Val Phe Gln Tyr 85 90 95Glu Asn Trp Asp Leu Phe Ile Glu Arg Ser Ser Ala Phe Ser Ser Cys 100 105110 Tyr Pro Tyr Asp Ile Pro Asp Tyr Ala Ser Leu Arg Ser Ile Val Ala 115120 125 Ser Ser Gly Thr Leu Glu Phe Thr Ala Glu Gly Phe Thr Trp Thr Gly130 135 140 Val Thr Gln Asn Gly Arg Ser Gly Ser Cys Lys Arg Gly Ser AlaAsp 145 150 155 160 Ser Phe Phe Ser Arg Leu Asn Trp Leu Thr Glu Ser GlyAsn Ser Tyr 165 170 175 Pro Thr Leu Asn Val Thr Met Pro Asn Asn Lys AsnPhe Asp Lys Leu 180 185 190 Tyr Ile Trp Gly Ile His His Pro Ser Ser AsnLys Glu Gln Thr Lys 195 200 205 Leu Tyr Ile Gln Glu Ser Gly Arg Val ThrVal Ser Thr Lys Arg Ser 210 215 220 Gln Gln Thr Ile Ile Pro Asn Ile GlySer Arg Pro Arg Val Arg Gly 225 230 235 240 Gln Ser Gly Arg Ile Ser IleTyr Trp Thr Ile Val Lys Pro Gly Asp 245 250 255 Ile Leu Met Ile Asn SerAsn Gly Asn Leu Val Ala Pro Arg Gly Tyr 260 265 270 Phe Lys Leu Lys ThrGly Lys Ser Ser Val Met Arg Ser Asp Ala Pro 275 280 285 Ile Asp Ile CysVal Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile Pro 290 295 300 Asn Asp LysPro Phe Gln Asn Val Asn Lys Val Thr Tyr Gly Lys Cys 305 310 315 320 ProLys Tyr Ile Arg Gln Asn Thr Leu Lys Leu Ala Thr Gly Met Arg 325 330 335Asn Val Pro Glu Lys Gln Ile Arg Gly Ile Phe Gly Ala Ile Ala Gly 340 345350 Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly Phe 355360 365 Arg Tyr Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser370 375 380 Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg ValIle 385 390 395 400 Glu Arg Thr Asn Glu Lys Phe His Gln Ile Glu Lys GluPhe Ser Glu 405 410 415 Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr ValGlu Asp Thr Lys 420 425 430 Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu LeuVal Ala Leu Lys Asn 435 440 445 Gln His Thr Ile Asp Leu Thr Asp Ala GluMet Asn Lys Leu Phe Glu 450 455 460 Lys Thr Arg Arg Gln Leu Arg Glu AsnAla Glu Asp Met Gly Gly Gly 465 470 475 480 Cys Phe Lys Ile Tyr His LysCys Asp Asn Ala Cys Ile Gly Ser Ile 485 490 495 Arg Asn Gly Thr Tyr AspHis Tyr Ile Tyr Arg Asp Glu Ala Leu Asn 500 505 510 Asn Arg Phe Gln IleLys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp 515 520 525 Trp Ile Leu TrpIle Ser Phe Ala Ile Ser Cys Phe Leu Ile Cys Val 530 535 540 Val Leu LeuGly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile Arg 545 550 555 560 CysAsn Ile Cys Ile 565 9 1695 DNA Equine influenza virus H3N8 9 atgaagacaaccattatttt gataccactg acccattggg tctacagtca aaacccaacc 60 agtggcaacaacacagccac attatgtctg ggacaccatg cagtagcaaa tggaacattg 120 gtaaaaacaataactgatga ccaaattgag gtgacaaatg ctactgaatt agttcagagc 180 atttcaatagggaaaatatg caacaactca tatagagttc tagatggaag aaattgcaca 240 ttaatagatgcaatgctagg agacccccac tgtgatgtct ttcagtatga gaattgggac 300 ctcttcatagaaagaagcag cgctttcagc agttgctacc catatgacat ccctgactat 360 gcatcgctccggtccattgt agcatcctca ggaacattgg aattcacagc agagggattc 420 acatggacaggtgtcactca aaacggaaga agtggatcct gcaaaagggg atcagccgat 480 agtttctttagccgactgaa ttggctaaca gaatctggaa actcttaccc cacattgaat 540 gtgacaatgcctaacaataa aaatttcgac aaactataca tctgggggat tcatcacccg 600 agctcaaacaaagagcagac aaaattgtac atccaagaat cgggacgagt aacagtctca 660 acaaaaagaagtcaacaaac aataatccct aacatcggat ctagaccgcg ggtcaggggt 720 caatcaggcaggataagcat atactggacc attgtaaaac ctggagatat cctaatgata 780 aacagtaatggcaacttagt tgcaccgcgg ggatatttta aattgaaaac agggaaaagc 840 tctgtaatgagatcagatgc acccatagac atttgtgtgt ctgaatgtat tacaccaaat 900 ggaagcatccccaacgacaa accatttcaa aatgtgaaca aagttacata tggaaaatgc 960 cccaagtatatcaggcaaaa cactttaaag ctggccactg ggatgaggaa tgtaccagaa 1020 aagcaaatcagaggaatctt tggagcaata gcgggattca tagaaaacgg ctgggaagga 1080 atggttgatgggtggtatgg attccgatat caaaactcgg aaggaacagg acaagctgca 1140 gatctaaagagcactcaagc agccatcgac cagatcaatg gaaaattaaa cagagtgatt 1200 gaaaggaccaatgagaaatt ccatcaaata gagaaggaat tctcagaagt agaagggagg 1260 atccaggacttggagaagta tgtagaagac accaaaatag acctatggtc ctacaatgca 1320 gaattgctggtggctctaaa aaatcaacat acaattgact taacagatgc agaaatgaat 1380 aaattattcgagaagactag acgccagtta agagaaaacg cggaagacat gggaggtgga 1440 tgtttcaagatataccacaa atgtgataat gcatgcattg gatcaataag aaatgggaca 1500 tatgaccattacatatacag agatgaagca ttaaacaacc ggtttcaaat caaaggtgtt 1560 gagttgaaatcaggctacaa agattggata ctgtggattt cattcgccat atcatgcttc 1620 ttaatttgcgttgttctatt gggtttcatt atgtgggctt gccaaaaagg caacatcaga 1680 tgcaacatttgcatt 1695 10 1762 DNA Equine influenza virus H3N8 CDS (30)..(1724) 10agcaaaagca ggggatattt ctgtcaatc atg aag aca acc att att ttg ata 53 MetLys Thr Thr Ile Ile Leu Ile 1 5 cta ctg acc cat tgg gtc tac agt caa aaccca acc agt ggc aac aac 101 Leu Leu Thr His Trp Val Tyr Ser Gln Asn ProThr Ser Gly Asn Asn 10 15 20 aca gcc aca tta tgt ctg gga cac cat gca gtagca aat gga aca ttg 149 Thr Ala Thr Leu Cys Leu Gly His His Ala Val AlaAsn Gly Thr Leu 25 30 35 40 gta aaa aca ata act gat gac caa att gag gtgaca aat gct act gaa 197 Val Lys Thr Ile Thr Asp Asp Gln Ile Glu Val ThrAsn Ala Thr Glu 45 50 55 tta gtt cag agc att tca ata ggg aaa ata tgc aacaac tca tat aga 245 Leu Val Gln Ser Ile Ser Ile Gly Lys Ile Cys Asn AsnSer Tyr Arg 60 65 70 gtt cta gat gga aga aat tgc aca tta ata gat gca atgcta gga gac 293 Val Leu Asp Gly Arg Asn Cys Thr Leu Ile Asp Ala Met LeuGly Asp 75 80 85 ccc cac tgt gat gtc ttt cag tat gag aat tgg gac ctc ttcata gaa 341 Pro His Cys Asp Val Phe Gln Tyr Glu Asn Trp Asp Leu Phe IleGlu 90 95 100 aga agc agc gct ttc agc agt tgc tac cca tat gac atc cctgac tat 389 Arg Ser Ser Ala Phe Ser Ser Cys Tyr Pro Tyr Asp Ile Pro AspTyr 105 110 115 120 gca tcg ctc cgg tcc att gta gca tcc tca gga aca ttggaa ttc aca 437 Ala Ser Leu Arg Ser Ile Val Ala Ser Ser Gly Thr Leu GluPhe Thr 125 130 135 gca gag gga ttc aca tgg aca ggt gtc act caa aac ggaaga agt gga 485 Ala Glu Gly Phe Thr Trp Thr Gly Val Thr Gln Asn Gly ArgSer Gly 140 145 150 tcc tgc aaa agg gaa tca gcc gat agt ttc ttt agc cgactg aat tgg 533 Ser Cys Lys Arg Glu Ser Ala Asp Ser Phe Phe Ser Arg LeuAsn Trp 155 160 165 cta aca gaa tct gga aac tct tac ccc aca ttg aat gtgaca atg cct 581 Leu Thr Glu Ser Gly Asn Ser Tyr Pro Thr Leu Asn Val ThrMet Pro 170 175 180 aac aat aaa aat ttc gac aaa cta tac atc tgg ggg attcat cac ccg 629 Asn Asn Lys Asn Phe Asp Lys Leu Tyr Ile Trp Gly Ile HisHis Pro 185 190 195 200 agc tca aac aaa gag cag aca aaa ttg tac atc caagaa tca gga cga 677 Ser Ser Asn Lys Glu Gln Thr Lys Leu Tyr Ile Gln GluSer Gly Arg 205 210 215 gta aca gtc tca aca aaa aga agt caa caa aca ataatc cct aac atc 725 Val Thr Val Ser Thr Lys Arg Ser Gln Gln Thr Ile IlePro Asn Ile 220 225 230 gga tct aga ccg tgg gtc agg ggt caa tca ggc aggata agc ata tac 773 Gly Ser Arg Pro Trp Val Arg Gly Gln Ser Gly Arg IleSer Ile Tyr 235 240 245 tgg acc att gta aaa cct gga gat atc cta acg ataaac agt aat ggc 821 Trp Thr Ile Val Lys Pro Gly Asp Ile Leu Thr Ile AsnSer Asn Gly 250 255 260 aac tta gtt gca ccg cgg gga tat ttt aaa ttg aaaaca ggg aaa agc 869 Asn Leu Val Ala Pro Arg Gly Tyr Phe Lys Leu Lys ThrGly Lys Ser 265 270 275 280 tct gta atg aga tca gat gca ccc ata gac atttgt gtg tct gaa tgt 917 Ser Val Met Arg Ser Asp Ala Pro Ile Asp Ile CysVal Ser Glu Cys 285 290 295 att aca cca aat gga agc atc ccc aac gac aaacca ttt caa aat gtg 965 Ile Thr Pro Asn Gly Ser Ile Pro Asn Asp Lys ProPhe Gln Asn Val 300 305 310 aac aaa gtt aca tat gga aaa tgc ccc aag tatatc agg caa aac act 1013 Asn Lys Val Thr Tyr Gly Lys Cys Pro Lys Tyr IleArg Gln Asn Thr 315 320 325 tta aag ctg gcc act ggg atg agg aat gta ccagaa aag caa atc aga 1061 Leu Lys Leu Ala Thr Gly Met Arg Asn Val Pro GluLys Gln Ile Arg 330 335 340 gga atc ttt gga gca ata gcg gga ttc ata gaaaac ggc tgg gaa gga 1109 Gly Ile Phe Gly Ala Ile Ala Gly Phe Ile Glu AsnGly Trp Glu Gly 345 350 355 360 atg gtt gat ggg tgg tat gga ttc cga tatcaa aac tcg gaa gga aca 1157 Met Val Asp Gly Trp Tyr Gly Phe Arg Tyr GlnAsn Ser Glu Gly Thr 365 370 375 gga caa gct gca gat cta aag agc act caagca gcc atc gac cag atc 1205 Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln AlaAla Ile Asp Gln Ile 380 385 390 aat gga aaa tta aac aga gtg att gaa aggacc aat gag aaa ttc cat 1253 Asn Gly Lys Leu Asn Arg Val Ile Glu Arg ThrAsn Glu Lys Phe His 395 400 405 caa ata gag aag gaa ttc tca gaa gta gaaggg aga atc cag gac ttg 1301 Gln Ile Glu Lys Glu Phe Ser Glu Val Glu GlyArg Ile Gln Asp Leu 410 415 420 gag aag tat gta gaa gac acc aaa ata gaccta tgg tcc tac aat gca 1349 Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp LeuTrp Ser Tyr Asn Ala 425 430 435 440 gaa ttg ctg gtg gct cta gaa aat caacat aca att gac tta aca gat 1397 Glu Leu Leu Val Ala Leu Glu Asn Gln HisThr Ile Asp Leu Thr Asp 445 450 455 gca gaa atg aat aaa tta ttc gag aagact aga cgc cag tta aga gaa 1445 Ala Glu Met Asn Lys Leu Phe Glu Lys ThrArg Arg Gln Leu Arg Glu 460 465 470 aac gcg gaa gac atg gga ggt gga tgtttc aag ata tac cac aaa tgt 1493 Asn Ala Glu Asp Met Gly Gly Gly Cys PheLys Ile Tyr His Lys Cys 475 480 485 gat aat gca tgc att gga tca ata agaaat ggg aca tat gac cat tac 1541 Asp Asn Ala Cys Ile Gly Ser Ile Arg AsnGly Thr Tyr Asp His Tyr 490 495 500 ata tac aga gat gaa gca tta aac aaccgg ttt caa atc aaa ggt gtt 1589 Ile Tyr Arg Asp Glu Ala Leu Asn Asn ArgPhe Gln Ile Lys Gly Val 505 510 515 520 gag ttg aaa tca ggc tac aaa gattgg ata ctg tgg att tca ttc gcc 1637 Glu Leu Lys Ser Gly Tyr Lys Asp TrpIle Leu Trp Ile Ser Phe Ala 525 530 535 ata tca tgc ttc tta att tgc gttgtt cta ttg ggt ttc att atg tgg 1685 Ile Ser Cys Phe Leu Ile Cys Val ValLeu Leu Gly Phe Ile Met Trp 540 545 550 gct tgc caa aaa ggc aac atc agatgc aac att tgc att tgagtaaact 1734 Ala Cys Gln Lys Gly Asn Ile Arg CysAsn Ile Cys Ile 555 560 565 gatagttaaa aacacccttg tttctact 1762 11 565PRT Equine influenza virus H3N8 11 Met Lys Thr Thr Ile Ile Leu Ile LeuLeu Thr His Trp Val Tyr Ser 1 5 10 15 Gln Asn Pro Thr Ser Gly Asn AsnThr Ala Thr Leu Cys Leu Gly His 20 25 30 His Ala Val Ala Asn Gly Thr LeuVal Lys Thr Ile Thr Asp Asp Gln 35 40 45 Ile Glu Val Thr Asn Ala Thr GluLeu Val Gln Ser Ile Ser Ile Gly 50 55 60 Lys Ile Cys Asn Asn Ser Tyr ArgVal Leu Asp Gly Arg Asn Cys Thr 65 70 75 80 Leu Ile Asp Ala Met Leu GlyAsp Pro His Cys Asp Val Phe Gln Tyr 85 90 95 Glu Asn Trp Asp Leu Phe IleGlu Arg Ser Ser Ala Phe Ser Ser Cys 100 105 110 Tyr Pro Tyr Asp Ile ProAsp Tyr Ala Ser Leu Arg Ser Ile Val Ala 115 120 125 Ser Ser Gly Thr LeuGlu Phe Thr Ala Glu Gly Phe Thr Trp Thr Gly 130 135 140 Val Thr Gln AsnGly Arg Ser Gly Ser Cys Lys Arg Glu Ser Ala Asp 145 150 155 160 Ser PhePhe Ser Arg Leu Asn Trp Leu Thr Glu Ser Gly Asn Ser Tyr 165 170 175 ProThr Leu Asn Val Thr Met Pro Asn Asn Lys Asn Phe Asp Lys Leu 180 185 190Tyr Ile Trp Gly Ile His His Pro Ser Ser Asn Lys Glu Gln Thr Lys 195 200205 Leu Tyr Ile Gln Glu Ser Gly Arg Val Thr Val Ser Thr Lys Arg Ser 210215 220 Gln Gln Thr Ile Ile Pro Asn Ile Gly Ser Arg Pro Trp Val Arg Gly225 230 235 240 Gln Ser Gly Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys ProGly Asp 245 250 255 Ile Leu Thr Ile Asn Ser Asn Gly Asn Leu Val Ala ProArg Gly Tyr 260 265 270 Phe Lys Leu Lys Thr Gly Lys Ser Ser Val Met ArgSer Asp Ala Pro 275 280 285 Ile Asp Ile Cys Val Ser Glu Cys Ile Thr ProAsn Gly Ser Ile Pro 290 295 300 Asn Asp Lys Pro Phe Gln Asn Val Asn LysVal Thr Tyr Gly Lys Cys 305 310 315 320 Pro Lys Tyr Ile Arg Gln Asn ThrLeu Lys Leu Ala Thr Gly Met Arg 325 330 335 Asn Val Pro Glu Lys Gln IleArg Gly Ile Phe Gly Ala Ile Ala Gly 340 345 350 Phe Ile Glu Asn Gly TrpGlu Gly Met Val Asp Gly Trp Tyr Gly Phe 355 360 365 Arg Tyr Gln Asn SerGlu Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser 370 375 380 Thr Gln Ala AlaIle Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile 385 390 395 400 Glu ArgThr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser Glu 405 410 415 ValGlu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys 420 425 430Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn 435 440445 Gln His Thr Ile Asp Leu Thr Asp Ala Glu Met Asn Lys Leu Phe Glu 450455 460 Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Gly Gly465 470 475 480 Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile GlySer Ile 485 490 495 Arg Asn Gly Thr Tyr Asp His Tyr Ile Tyr Arg Asp GluAla Leu Asn 500 505 510 Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys SerGly Tyr Lys Asp 515 520 525 Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser CysPhe Leu Ile Cys Val 530 535 540 Val Leu Leu Gly Phe Ile Met Trp Ala CysGln Lys Gly Asn Ile Arg 545 550 555 560 Cys Asn Ile Cys Ile 565 12 1695DNA Equine influenza virus H3N8 12 atgaagacaa ccattatttt gatactactgacccattggg tctacagtca aaacccaacc 60 agtggcaaca acacagccac attatgtctgggacaccatg cagtagcaaa tggaacattg 120 gtaaaaacaa taactgatga ccaaattgaggtgacaaatg ctactgaatt agttcagagc 180 atttcaatag ggaaaatatg caacaactcatatagagttc tagatggaag aaattgcaca 240 ttaatagatg caatgctagg agacccccactgtgatgtct ttcagtatga gaattgggac 300 ctcttcatag aaagaagcag cgctttcagcagttgctacc catatgacat ccctgactat 360 gcatcgctcc ggtccattgt agcatcctcaggaacattgg aattcacagc agagggattc 420 acatggacag gtgtcactca aaacggaagaagtggatcct gcaaaaggga atcagccgat 480 agtttcttta gccgactgaa ttggctaacagaatctggaa actcttaccc cacattgaat 540 gtgacaatgc ctaacaataa aaatttcgacaaactataca tctgggggat tcatcacccg 600 agctcaaaca aagagcagac aaaattgtacatccaagaat caggacgagt aacagtctca 660 acaaaaagaa gtcaacaaac aataatccctaacatcggat ctagaccgtg ggtcaggggt 720 caatcaggca ggataagcat atactggaccattgtaaaac ctggagatat cctaacgata 780 aacagtaatg gcaacttagt tgcaccgcggggatatttta aattgaaaac agggaaaagc 840 tctgtaatga gatcagatgc acccatagacatttgtgtgt ctgaatgtat tacaccaaat 900 ggaagcatcc ccaacgacaa accatttcaaaatgtgaaca aagttacata tggaaaatgc 960 cccaagtata tcaggcaaaa cactttaaagctggccactg ggatgaggaa tgtaccagaa 1020 aagcaaatca gaggaatctt tggagcaatagcgggattca tagaaaacgg ctgggaagga 1080 atggttgatg ggtggtatgg attccgatatcaaaactcgg aaggaacagg acaagctgca 1140 gatctaaaga gcactcaagc agccatcgaccagatcaatg gaaaattaaa cagagtgatt 1200 gaaaggacca atgagaaatt ccatcaaatagagaaggaat tctcagaagt agaagggaga 1260 atccaggact tggagaagta tgtagaagacaccaaaatag acctatggtc ctacaatgca 1320 gaattgctgg tggctctaga aaatcaacatacaattgact taacagatgc agaaatgaat 1380 aaattattcg agaagactag acgccagttaagagaaaacg cggaagacat gggaggtgga 1440 tgtttcaaga tataccacaa atgtgataatgcatgcattg gatcaataag aaatgggaca 1500 tatgaccatt acatatacag agatgaagcattaaacaacc ggtttcaaat caaaggtgtt 1560 gagttgaaat caggctacaa agattggatactgtggattt cattcgccat atcatgcttc 1620 ttaatttgcg ttgttctatt gggtttcattatgtgggctt gccaaaaagg caacatcaga 1680 tgcaacattt gcatt 1695 13 1241 DNAEquine influenza virus H3N8 CDS (28)..(1239) 13 agcaaaagca ggtcaaatatattcaat atg gag aga ata aaa gaa ctg aga gat 54 Met Glu Arg Ile Lys GluLeu Arg Asp 1 5 cta atg tca caa tcc cgc acc cgc gag ata cta aca aaa actact gtg 102 Leu Met Ser Gln Ser Arg Thr Arg Glu Ile Leu Thr Lys Thr ThrVal 10 15 20 25 gac cac atg gcc ata atc aag aaa tac aca tca gga aga caagag aag 150 Asp His Met Ala Ile Ile Lys Lys Tyr Thr Ser Gly Arg Gln GluLys 30 35 40 aac ccc gca ctt agg atg aag tgg atg atg gca atg aaa tac ccaatt 198 Asn Pro Ala Leu Arg Met Lys Trp Met Met Ala Met Lys Tyr Pro Ile45 50 55 aca gca gat aag agg ata atg gaa atg att cct gag aga aat gaa cag246 Thr Ala Asp Lys Arg Ile Met Glu Met Ile Pro Glu Arg Asn Glu Gln 6065 70 ggg caa acc ctt tgg agc aaa acg aac gat gct ggc tca gac cgc gta294 Gly Gln Thr Leu Trp Ser Lys Thr Asn Asp Ala Gly Ser Asp Arg Val 7580 85 atg gta tca cct ctg gca gtg aca tgg tgg aat agg aat gga cca aca342 Met Val Ser Pro Leu Ala Val Thr Trp Trp Asn Arg Asn Gly Pro Thr 9095 100 105 acg agc aca att cat tat cca aaa gtc tac aaa act tat ttt gaaaaa 390 Thr Ser Thr Ile His Tyr Pro Lys Val Tyr Lys Thr Tyr Phe Glu Lys110 115 120 gtt gaa aga tta aaa cac gga acc ttt ggc ccc gtt cat ttt aggaat 438 Val Glu Arg Leu Lys His Gly Thr Phe Gly Pro Val His Phe Arg Asn125 130 135 caa gtc aag ata aga cgg aga gtt gat gta aac cct ggt cac gcggac 486 Gln Val Lys Ile Arg Arg Arg Val Asp Val Asn Pro Gly His Ala Asp140 145 150 ctc agt gcc aaa gaa gca caa gat gtg atc atg gaa gtt gtt ttccca 534 Leu Ser Ala Lys Glu Ala Gln Asp Val Ile Met Glu Val Val Phe Pro155 160 165 aat gaa gtg gga gcc aga att cta aca tcg gaa tca caa cta acaata 582 Asn Glu Val Gly Ala Arg Ile Leu Thr Ser Glu Ser Gln Leu Thr Ile170 175 180 185 acc aaa gag aaa aaa gaa gaa ctt cag gac tgc aaa att gccccc ttg 630 Thr Lys Glu Lys Lys Glu Glu Leu Gln Asp Cys Lys Ile Ala ProLeu 190 195 200 atg gta gca tac atg cta gaa aga gag ttg gtc cga aaa acaaga ttc 678 Met Val Ala Tyr Met Leu Glu Arg Glu Leu Val Arg Lys Thr ArgPhe 205 210 215 ctc cca gtg gct ggc gga aca agc agt gta tac att gaa gtgttg cat 726 Leu Pro Val Ala Gly Gly Thr Ser Ser Val Tyr Ile Glu Val LeuHis 220 225 230 ctg act cag gga aca tgc tgg gaa caa atg tac acc cca ggagga gaa 774 Leu Thr Gln Gly Thr Cys Trp Glu Gln Met Tyr Thr Pro Gly GlyGlu 235 240 245 gtt aga aac gat gac att gat caa agt tta att att gct gcccgg aac 822 Val Arg Asn Asp Asp Ile Asp Gln Ser Leu Ile Ile Ala Ala ArgAsn 250 255 260 265 ata gtg aga aga gcg aca gta tca gca gat cca cta gcatcc ctg ctg 870 Ile Val Arg Arg Ala Thr Val Ser Ala Asp Pro Leu Ala SerLeu Leu 270 275 280 gaa atg tgc cac agt aca cag att ggt gga ata agg atggta gac atc 918 Glu Met Cys His Ser Thr Gln Ile Gly Gly Ile Arg Met ValAsp Ile 285 290 295 ctt aag cag aat cca aca gag gaa caa gct gtg gat atatgc aaa gca 966 Leu Lys Gln Asn Pro Thr Glu Glu Gln Ala Val Asp Ile CysLys Ala 300 305 310 gca atg ggg tta aga att agc tca tca ttc agc ttt ggtgga ttc acc 1014 Ala Met Gly Leu Arg Ile Ser Ser Ser Phe Ser Phe Gly GlyPhe Thr 315 320 325 ttt aag aga aca agt gga tca tca gtc aag aga gaa gaagaa atg ctt 1062 Phe Lys Arg Thr Ser Gly Ser Ser Val Lys Arg Glu Glu GluMet Leu 330 335 340 345 acg ggc aac ctt caa aca ttg aaa ata aga gtg catgaa ggc tat gaa 1110 Thr Gly Asn Leu Gln Thr Leu Lys Ile Arg Val His GluGly Tyr Glu 350 355 360 gaa ttc aca atg gtc gga aga aga gca aca gcc attctc aga aag gca 1158 Glu Phe Thr Met Val Gly Arg Arg Ala Thr Ala Ile LeuArg Lys Ala 365 370 375 acc aga aga ttg att caa ttg ata gta agt ggg agagat gaa caa tca 1206 Thr Arg Arg Leu Ile Gln Leu Ile Val Ser Gly Arg AspGlu Gln Ser 380 385 390 att gct gaa gca ata att gta gcc atg gtg ttt tc1241 Ile Ala Glu Ala Ile Ile Val Ala Met Val Phe 395 400 14 404 PRTEquine influenza virus H3N8 14 Met Glu Arg Ile Lys Glu Leu Arg Asp LeuMet Ser Gln Ser Arg Thr 1 5 10 15 Arg Glu Ile Leu Thr Lys Thr Thr ValAsp His Met Ala Ile Ile Lys 20 25 30 Lys Tyr Thr Ser Gly Arg Gln Glu LysAsn Pro Ala Leu Arg Met Lys 35 40 45 Trp Met Met Ala Met Lys Tyr Pro IleThr Ala Asp Lys Arg Ile Met 50 55 60 Glu Met Ile Pro Glu Arg Asn Glu GlnGly Gln Thr Leu Trp Ser Lys 65 70 75 80 Thr Asn Asp Ala Gly Ser Asp ArgVal Met Val Ser Pro Leu Ala Val 85 90 95 Thr Trp Trp Asn Arg Asn Gly ProThr Thr Ser Thr Ile His Tyr Pro 100 105 110 Lys Val Tyr Lys Thr Tyr PheGlu Lys Val Glu Arg Leu Lys His Gly 115 120 125 Thr Phe Gly Pro Val HisPhe Arg Asn Gln Val Lys Ile Arg Arg Arg 130 135 140 Val Asp Val Asn ProGly His Ala Asp Leu Ser Ala Lys Glu Ala Gln 145 150 155 160 Asp Val IleMet Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg Ile 165 170 175 Leu ThrSer Glu Ser Gln Leu Thr Ile Thr Lys Glu Lys Lys Glu Glu 180 185 190 LeuGln Asp Cys Lys Ile Ala Pro Leu Met Val Ala Tyr Met Leu Glu 195 200 205Arg Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Ala Gly Gly Thr 210 215220 Ser Ser Val Tyr Ile Glu Val Leu His Leu Thr Gln Gly Thr Cys Trp 225230 235 240 Glu Gln Met Tyr Thr Pro Gly Gly Glu Val Arg Asn Asp Asp IleAsp 245 250 255 Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg AlaThr Val 260 265 270 Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys HisSer Thr Gln 275 280 285 Ile Gly Gly Ile Arg Met Val Asp Ile Leu Lys GlnAsn Pro Thr Glu 290 295 300 Glu Gln Ala Val Asp Ile Cys Lys Ala Ala MetGly Leu Arg Ile Ser 305 310 315 320 Ser Ser Phe Ser Phe Gly Gly Phe ThrPhe Lys Arg Thr Ser Gly Ser 325 330 335 Ser Val Lys Arg Glu Glu Glu MetLeu Thr Gly Asn Leu Gln Thr Leu 340 345 350 Lys Ile Arg Val His Glu GlyTyr Glu Glu Phe Thr Met Val Gly Arg 355 360 365 Arg Ala Thr Ala Ile LeuArg Lys Ala Thr Arg Arg Leu Ile Gln Leu 370 375 380 Ile Val Ser Gly ArgAsp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val 385 390 395 400 Ala Met ValPhe 15 1214 DNA Equine influenza virus H3N8 15 atggagagaa taaaagaactgagagatcta atgtcacaat cccgcacccg cgagatacta 60 acaaaaacta ctgtggaccacatggccata atcaagaaat acacatcagg aagacaagag 120 aagaaccccg cacttaggatgaagtggatg atggcaatga aatacccaat tacagcagat 180 aagaggataa tggaaatgattcctgagaga aatgaacagg ggcaaaccct ttggagcaaa 240 acgaacgatg ctggctcagaccgcgtaatg gtatcacctc tggcagtgac atggtggaat 300 aggaatggac caacaacgagcacaattcat tatccaaaag tctacaaaac ttattttgaa 360 aaagttgaaa gattaaaacacggaaccttt ggccccgttc attttaggaa tcaagtcaag 420 ataagacgga gagttgatgtaaaccctggt cacgcggacc tcagtgccaa agaagcacaa 480 gatgtgatca tggaagttgttttcccaaat gaagtgggag ccagaattct aacatcggaa 540 tcacaactaa caataaccaaagagaaaaaa gaagaacttc aggactgcaa aattgccccc 600 ttgatggtag catacatgctagaaagagag ttggtccgaa aaacaagatt cctcccagtg 660 gctggcggaa caagcagtgtatacattgaa gtgttgcatc tgactcaggg aacatgctgg 720 gaacaaatgt acaccccaggaggagaagtt agaaacgatg acattgatca aagtttaatt 780 attgctgccc ggaacatagtgagaagagcg acagtatcag cagatccact agcatccctg 840 ctggaaatgt gccacagtacacagattggt ggaataagga tggtagacat ccttaagcag 900 aatccaacag aggaacaagctgtggatata tgcaaagcag caatggggtt aagaattagc 960 tcatcattca gctttggtggattcaccttt aagagaacaa gtggatcatc agtcaagaga 1020 gaagaagaaa tgcttacgggcaaccttcaa acattgaaaa taagagtgca tgaaggctat 1080 gaagaattca caatggtcggaagaagagca acagccattc tcagaaaggc aaccagaaga 1140 ttgattcaat tgatagtaagtgggagagat gaacaatcaa ttgctgaagc aataattgta 1200 gccatggtgt tttc 1214 161241 DNA Equine influenza virus H3N8 CDS (28)..(1239) 16 agcaaaagcaggtcaaatat attcaat atg gag aga ata aaa gaa ctg aga gat 54 Met Glu ArgIle Lys Glu Leu Arg Asp 1 5 cta atg tca caa tcc cgc acc cgc gag ata ctaaca aaa act act gtg 102 Leu Met Ser Gln Ser Arg Thr Arg Glu Ile Leu ThrLys Thr Thr Val 10 15 20 25 gac cac atg gcc ata atc aag aaa tac aca tcagga aga caa gag aag 150 Asp His Met Ala Ile Ile Lys Lys Tyr Thr Ser GlyArg Gln Glu Lys 30 35 40 aac ccc gca ctt agg atg aag tgg atg atg gca atgaaa tac cca att 198 Asn Pro Ala Leu Arg Met Lys Trp Met Met Ala Met LysTyr Pro Ile 45 50 55 aca gca gat aag agg ata atg gaa atg att cct gag agaaat gaa cag 246 Thr Ala Asp Lys Arg Ile Met Glu Met Ile Pro Glu Arg AsnGlu Gln 60 65 70 ggg caa acc ctt tgg agc aaa acg aac gat gct ggc tca gaccgc gta 294 Gly Gln Thr Leu Trp Ser Lys Thr Asn Asp Ala Gly Ser Asp ArgVal 75 80 85 atg gta tca cct ctg gca gtg aca tgg tgg aat agg aat gga ccaaca 342 Met Val Ser Pro Leu Ala Val Thr Trp Trp Asn Arg Asn Gly Pro Thr90 95 100 105 acg agc aca att cat tat cca aaa gtc cac aaa act tat tttgaa aaa 390 Thr Ser Thr Ile His Tyr Pro Lys Val His Lys Thr Tyr Phe GluLys 110 115 120 gtt gaa aga tta aaa cac gga acc ttt ggc ccc gtt cat tttagg aat 438 Val Glu Arg Leu Lys His Gly Thr Phe Gly Pro Val His Phe ArgAsn 125 130 135 caa gtc aag ata aga cgg aga gtt gat gta aac cct ggt cacgcg gac 486 Gln Val Lys Ile Arg Arg Arg Val Asp Val Asn Pro Gly His AlaAsp 140 145 150 ctc agt gcc aaa gaa gca caa gat gtg atc atg gaa gtt gttttc cca 534 Leu Ser Ala Lys Glu Ala Gln Asp Val Ile Met Glu Val Val PhePro 155 160 165 aat gaa gtg gga gcc aga att cta aca tcg gaa tca caa ctaaca ata 582 Asn Glu Val Gly Ala Arg Ile Leu Thr Ser Glu Ser Gln Leu ThrIle 170 175 180 185 acc aaa gag aaa aaa gaa gaa ctt cag gac tgc aaa attgcc ccc ttg 630 Thr Lys Glu Lys Lys Glu Glu Leu Gln Asp Cys Lys Ile AlaPro Leu 190 195 200 atg gta gca tac atg cta gaa aga gag ttg gtc cga aaaaca aga ttc 678 Met Val Ala Tyr Met Leu Glu Arg Glu Leu Val Arg Lys ThrArg Phe 205 210 215 ctc cca gtg gct ggc gga aca agc agt gta tac att gaagtg ttg cat 726 Leu Pro Val Ala Gly Gly Thr Ser Ser Val Tyr Ile Glu ValLeu His 220 225 230 ctg act cag gga aca tgc tgg gaa caa atg tac acc ccagga gga gaa 774 Leu Thr Gln Gly Thr Cys Trp Glu Gln Met Tyr Thr Pro GlyGly Glu 235 240 245 gtt aga aac gat gac att gat caa agt tta att att gctgcc cgg aac 822 Val Arg Asn Asp Asp Ile Asp Gln Ser Leu Ile Ile Ala AlaArg Asn 250 255 260 265 ata gtg aga aga gcg aca gta tca gca gat cca ctagca tcc ctg ctg 870 Ile Val Arg Arg Ala Thr Val Ser Ala Asp Pro Leu AlaSer Leu Leu 270 275 280 gaa atg tgc cac agt aca cag att ggt gga ata aggatg gta gac atc 918 Glu Met Cys His Ser Thr Gln Ile Gly Gly Ile Arg MetVal Asp Ile 285 290 295 ctt aag cag aat cca aca gag gaa caa gct gtg gatata tgc aaa gca 966 Leu Lys Gln Asn Pro Thr Glu Glu Gln Ala Val Asp IleCys Lys Ala 300 305 310 gca atg ggg tta aga att agc tca tca ttc agc tttggt gga ttc acc 1014 Ala Met Gly Leu Arg Ile Ser Ser Ser Phe Ser Phe GlyGly Phe Thr 315 320 325 ttt aag aga aca agt gga tca tca gtc aag aga gaagaa gaa atg ctt 1062 Phe Lys Arg Thr Ser Gly Ser Ser Val Lys Arg Glu GluGlu Met Leu 330 335 340 345 acg ggc aac ctt caa aca ttg aaa ata aga gtgcat gaa ggc tat gaa 1110 Thr Gly Asn Leu Gln Thr Leu Lys Ile Arg Val HisGlu Gly Tyr Glu 350 355 360 gaa ttc aca atg gtc gga aga aga gca aca gccatt ctc aga aag gca 1158 Glu Phe Thr Met Val Gly Arg Arg Ala Thr Ala IleLeu Arg Lys Ala 365 370 375 acc aga aga ttg att caa ttg ata gta agt gggaga gat gaa caa tca 1206 Thr Arg Arg Leu Ile Gln Leu Ile Val Ser Gly ArgAsp Glu Gln Ser 380 385 390 att gct gaa gca ata att gta gcc atg gtg ttttc 1241 Ile Ala Glu Ala Ile Ile Val Ala Met Val Phe 395 400 17 404 PRTEquine influenza virus H3N8 17 Met Glu Arg Ile Lys Glu Leu Arg Asp LeuMet Ser Gln Ser Arg Thr 1 5 10 15 Arg Glu Ile Leu Thr Lys Thr Thr ValAsp His Met Ala Ile Ile Lys 20 25 30 Lys Tyr Thr Ser Gly Arg Gln Glu LysAsn Pro Ala Leu Arg Met Lys 35 40 45 Trp Met Met Ala Met Lys Tyr Pro IleThr Ala Asp Lys Arg Ile Met 50 55 60 Glu Met Ile Pro Glu Arg Asn Glu GlnGly Gln Thr Leu Trp Ser Lys 65 70 75 80 Thr Asn Asp Ala Gly Ser Asp ArgVal Met Val Ser Pro Leu Ala Val 85 90 95 Thr Trp Trp Asn Arg Asn Gly ProThr Thr Ser Thr Ile His Tyr Pro 100 105 110 Lys Val His Lys Thr Tyr PheGlu Lys Val Glu Arg Leu Lys His Gly 115 120 125 Thr Phe Gly Pro Val HisPhe Arg Asn Gln Val Lys Ile Arg Arg Arg 130 135 140 Val Asp Val Asn ProGly His Ala Asp Leu Ser Ala Lys Glu Ala Gln 145 150 155 160 Asp Val IleMet Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg Ile 165 170 175 Leu ThrSer Glu Ser Gln Leu Thr Ile Thr Lys Glu Lys Lys Glu Glu 180 185 190 LeuGln Asp Cys Lys Ile Ala Pro Leu Met Val Ala Tyr Met Leu Glu 195 200 205Arg Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Ala Gly Gly Thr 210 215220 Ser Ser Val Tyr Ile Glu Val Leu His Leu Thr Gln Gly Thr Cys Trp 225230 235 240 Glu Gln Met Tyr Thr Pro Gly Gly Glu Val Arg Asn Asp Asp IleAsp 245 250 255 Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg AlaThr Val 260 265 270 Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys HisSer Thr Gln 275 280 285 Ile Gly Gly Ile Arg Met Val Asp Ile Leu Lys GlnAsn Pro Thr Glu 290 295 300 Glu Gln Ala Val Asp Ile Cys Lys Ala Ala MetGly Leu Arg Ile Ser 305 310 315 320 Ser Ser Phe Ser Phe Gly Gly Phe ThrPhe Lys Arg Thr Ser Gly Ser 325 330 335 Ser Val Lys Arg Glu Glu Glu MetLeu Thr Gly Asn Leu Gln Thr Leu 340 345 350 Lys Ile Arg Val His Glu GlyTyr Glu Glu Phe Thr Met Val Gly Arg 355 360 365 Arg Ala Thr Ala Ile LeuArg Lys Ala Thr Arg Arg Leu Ile Gln Leu 370 375 380 Ile Val Ser Gly ArgAsp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val 385 390 395 400 Ala Met ValPhe 18 1214 DNA Equine influenza virus H3N8 18 atggagagaa taaaagaactgagagatcta atgtcacaat cccgcacccg cgagatacta 60 acaaaaacta ctgtggaccacatggccata atcaagaaat acacatcagg aagacaagag 120 aagaaccccg cacttaggatgaagtggatg atggcaatga aatacccaat tacagcagat 180 aagaggataa tggaaatgattcctgagaga aatgaacagg ggcaaaccct ttggagcaaa 240 acgaacgatg ctggctcagaccgcgtaatg gtatcacctc tggcagtgac atggtggaat 300 aggaatggac caacaacgagcacaattcat tatccaaaag tccacaaaac ttattttgaa 360 aaagttgaaa gattaaaacacggaaccttt ggccccgttc attttaggaa tcaagtcaag 420 ataagacgga gagttgatgtaaaccctggt cacgcggacc tcagtgccaa agaagcacaa 480 gatgtgatca tggaagttgttttcccaaat gaagtgggag ccagaattct aacatcggaa 540 tcacaactaa caataaccaaagagaaaaaa gaagaacttc aggactgcaa aattgccccc 600 ttgatggtag catacatgctagaaagagag ttggtccgaa aaacaagatt cctcccagtg 660 gctggcggaa caagcagtgtatacattgaa gtgttgcatc tgactcaggg aacatgctgg 720 gaacaaatgt acaccccaggaggagaagtt agaaacgatg acattgatca aagtttaatt 780 attgctgccc ggaacatagtgagaagagcg acagtatcag cagatccact agcatccctg 840 ctggaaatgt gccacagtacacagattggt ggaataagga tggtagacat ccttaagcag 900 aatccaacag aggaacaagctgtggatata tgcaaagcag caatggggtt aagaattagc 960 tcatcattca gctttggtggattcaccttt aagagaacaa gtggatcatc agtcaagaga 1020 gaagaagaaa tgcttacgggcaaccttcaa acattgaaaa taagagtgca tgaaggctat 1080 gaagaattca caatggtcggaagaagagca acagccattc tcagaaaggc aaccagaaga 1140 ttgattcaat tgatagtaagtgggagagat gaacaatcaa ttgctgaagc aataattgta 1200 gccatggtgt tttc 1214 191233 DNA Equine influenza virus H3N8 CDS (3)..(1196) 19 ta gaa ttc acaatg gtc gga aga aga gca aca gcc att ctc aga aag 47 Glu Phe Thr Met ValGly Arg Arg Ala Thr Ala Ile Leu Arg Lys 1 5 10 15 gca acc aga aga ttgatt caa ttg ata gta agt ggg aga gat gaa caa 95 Ala Thr Arg Arg Leu IleGln Leu Ile Val Ser Gly Arg Asp Glu Gln 20 25 30 tca att gct gaa gca ataatt gta gcc atg gtg ttt tcg caa gaa gat 143 Ser Ile Ala Glu Ala Ile IleVal Ala Met Val Phe Ser Gln Glu Asp 35 40 45 tgc atg ata aaa gca gtt cgaggc gat ttg aac ttc gtt aat aga gca 191 Cys Met Ile Lys Ala Val Arg GlyAsp Leu Asn Phe Val Asn Arg Ala 50 55 60 aat cag cgc ttg aac ccc atg catcaa ctc ttg agg cat ttc caa aaa 239 Asn Gln Arg Leu Asn Pro Met His GlnLeu Leu Arg His Phe Gln Lys 65 70 75 gat gca aaa gtg ctt ttc cag aat tggggg att gaa ccc atc gac aat 287 Asp Ala Lys Val Leu Phe Gln Asn Trp GlyIle Glu Pro Ile Asp Asn 80 85 90 95 gtg atg gga atg att gga ata ttg cctgac atg acc cca agc acc gag 335 Val Met Gly Met Ile Gly Ile Leu Pro AspMet Thr Pro Ser Thr Glu 100 105 110 atg tca ttg aga gga gtg aga gtc agcaaa atg gga gtg gat gag tac 383 Met Ser Leu Arg Gly Val Arg Val Ser LysMet Gly Val Asp Glu Tyr 115 120 125 tcc agc act gag aga gtg gtg gtg agcatt gac cgt ttt tta aga gtt 431 Ser Ser Thr Glu Arg Val Val Val Ser IleAsp Arg Phe Leu Arg Val 130 135 140 cgg gat caa agg gga aac ata cta ctgtcc cct gaa gag gtc agt gaa 479 Arg Asp Gln Arg Gly Asn Ile Leu Leu SerPro Glu Glu Val Ser Glu 145 150 155 aca caa gga acg gaa aag ctg aca ataatt tat tca tca tca atg atg 527 Thr Gln Gly Thr Glu Lys Leu Thr Ile IleTyr Ser Ser Ser Met Met 160 165 170 175 tgg gag att aat ggt ccc gaa tcagtg ttg gtc aat act tat caa tgg 575 Trp Glu Ile Asn Gly Pro Glu Ser ValLeu Val Asn Thr Tyr Gln Trp 180 185 190 atc atc agg aac tgg gaa att gtgaaa att caa tgg tca cag gat ccc 623 Ile Ile Arg Asn Trp Glu Ile Val LysIle Gln Trp Ser Gln Asp Pro 195 200 205 aca atg tta tac aat aag ata gaattt gag cca ttc cag tcc ctg gtc 671 Thr Met Leu Tyr Asn Lys Ile Glu PheGlu Pro Phe Gln Ser Leu Val 210 215 220 cct agg gcc acc aga agc caa tacagc ggt ttc gta aga acc ctg ttt 719 Pro Arg Ala Thr Arg Ser Gln Tyr SerGly Phe Val Arg Thr Leu Phe 225 230 235 cag caa atg cga gat gta ctt ggaaca ttt gat act gct caa ata ata 767 Gln Gln Met Arg Asp Val Leu Gly ThrPhe Asp Thr Ala Gln Ile Ile 240 245 250 255 aaa ctc ctc cct ttt gcc gctgct cct ccg gaa cag agt agg atg cag 815 Lys Leu Leu Pro Phe Ala Ala AlaPro Pro Glu Gln Ser Arg Met Gln 260 265 270 ttc tct tct ttg act gtt aatgta aga gga tcg gga atg agg ata ctt 863 Phe Ser Ser Leu Thr Val Asn ValArg Gly Ser Gly Met Arg Ile Leu 275 280 285 gta aga ggc aat tcc cca gtgttc aac tac aat aaa gcc act aag agg 911 Val Arg Gly Asn Ser Pro Val PheAsn Tyr Asn Lys Ala Thr Lys Arg 290 295 300 ctc aca gtc ctc gga aag gatgca ggt gcg ctt act gaa gac cca gat 959 Leu Thr Val Leu Gly Lys Asp AlaGly Ala Leu Thr Glu Asp Pro Asp 305 310 315 gaa ggt acg gct gga gta gaatct gct gtt cta aga ggg ttt ctc att 1007 Glu Gly Thr Ala Gly Val Glu SerAla Val Leu Arg Gly Phe Leu Ile 320 325 330 335 tta ggt aaa gaa aac aagaga tat ggc cca gca cta agc atc aat gaa 1055 Leu Gly Lys Glu Asn Lys ArgTyr Gly Pro Ala Leu Ser Ile Asn Glu 340 345 350 ctg agc aaa ctt gca aaaggg gag aaa gct aat gtg cta att ggg caa 1103 Leu Ser Lys Leu Ala Lys GlyGlu Lys Ala Asn Val Leu Ile Gly Gln 355 360 365 ggg gac gtg gtg ttg gtaatg aaa cgg aaa cgt gac tct agc ata ctt 1151 Gly Asp Val Val Leu Val MetLys Arg Lys Arg Asp Ser Ser Ile Leu 370 375 380 act gac agc cag aca gcgacc aaa agg att cgg atg gcc atc aat 1196 Thr Asp Ser Gln Thr Ala Thr LysArg Ile Arg Met Ala Ile Asn 385 390 395 tagtgttgaa ttgtttaaaa acgaccttgtttctact 1233 20 398 PRT Equine influenza virus H3N8 20 Glu Phe Thr MetVal Gly Arg Arg Ala Thr Ala Ile Leu Arg Lys Ala 1 5 10 15 Thr Arg ArgLeu Ile Gln Leu Ile Val Ser Gly Arg Asp Glu Gln Ser 20 25 30 Ile Ala GluAla Ile Ile Val Ala Met Val Phe Ser Gln Glu Asp Cys 35 40 45 Met Ile LysAla Val Arg Gly Asp Leu Asn Phe Val Asn Arg Ala Asn 50 55 60 Gln Arg LeuAsn Pro Met His Gln Leu Leu Arg His Phe Gln Lys Asp 65 70 75 80 Ala LysVal Leu Phe Gln Asn Trp Gly Ile Glu Pro Ile Asp Asn Val 85 90 95 Met GlyMet Ile Gly Ile Leu Pro Asp Met Thr Pro Ser Thr Glu Met 100 105 110 SerLeu Arg Gly Val Arg Val Ser Lys Met Gly Val Asp Glu Tyr Ser 115 120 125Ser Thr Glu Arg Val Val Val Ser Ile Asp Arg Phe Leu Arg Val Arg 130 135140 Asp Gln Arg Gly Asn Ile Leu Leu Ser Pro Glu Glu Val Ser Glu Thr 145150 155 160 Gln Gly Thr Glu Lys Leu Thr Ile Ile Tyr Ser Ser Ser Met MetTrp 165 170 175 Glu Ile Asn Gly Pro Glu Ser Val Leu Val Asn Thr Tyr GlnTrp Ile 180 185 190 Ile Arg Asn Trp Glu Ile Val Lys Ile Gln Trp Ser GlnAsp Pro Thr 195 200 205 Met Leu Tyr Asn Lys Ile Glu Phe Glu Pro Phe GlnSer Leu Val Pro 210 215 220 Arg Ala Thr Arg Ser Gln Tyr Ser Gly Phe ValArg Thr Leu Phe Gln 225 230 235 240 Gln Met Arg Asp Val Leu Gly Thr PheAsp Thr Ala Gln Ile Ile Lys 245 250 255 Leu Leu Pro Phe Ala Ala Ala ProPro Glu Gln Ser Arg Met Gln Phe 260 265 270 Ser Ser Leu Thr Val Asn ValArg Gly Ser Gly Met Arg Ile Leu Val 275 280 285 Arg Gly Asn Ser Pro ValPhe Asn Tyr Asn Lys Ala Thr Lys Arg Leu 290 295 300 Thr Val Leu Gly LysAsp Ala Gly Ala Leu Thr Glu Asp Pro Asp Glu 305 310 315 320 Gly Thr AlaGly Val Glu Ser Ala Val Leu Arg Gly Phe Leu Ile Leu 325 330 335 Gly LysGlu Asn Lys Arg Tyr Gly Pro Ala Leu Ser Ile Asn Glu Leu 340 345 350 SerLys Leu Ala Lys Gly Glu Lys Ala Asn Val Leu Ile Gly Gln Gly 355 360 365Asp Val Val Leu Val Met Lys Arg Lys Arg Asp Ser Ser Ile Leu Thr 370 375380 Asp Ser Gln Thr Ala Thr Lys Arg Ile Arg Met Ala Ile Asn 385 390 39521 1194 DNA Equine influenza virus H3N8 21 gaattcacaa tggtcggaagaagagcaaca gccattctca gaaaggcaac cagaagattg 60 attcaattga tagtaagtgggagagatgaa caatcaattg ctgaagcaat aattgtagcc 120 atggtgtttt cgcaagaagattgcatgata aaagcagttc gaggcgattt gaacttcgtt 180 aatagagcaa atcagcgcttgaaccccatg catcaactct tgaggcattt ccaaaaagat 240 gcaaaagtgc ttttccagaattgggggatt gaacccatcg acaatgtgat gggaatgatt 300 ggaatattgc ctgacatgaccccaagcacc gagatgtcat tgagaggagt gagagtcagc 360 aaaatgggag tggatgagtactccagcact gagagagtgg tggtgagcat tgaccgtttt 420 ttaagagttc gggatcaaaggggaaacata ctactgtccc ctgaagaggt cagtgaaaca 480 caaggaacgg aaaagctgacaataatttat tcatcatcaa tgatgtggga gattaatggt 540 cccgaatcag tgttggtcaatacttatcaa tggatcatca ggaactggga aattgtgaaa 600 attcaatggt cacaggatcccacaatgtta tacaataaga tagaatttga gccattccag 660 tccctggtcc ctagggccaccagaagccaa tacagcggtt tcgtaagaac cctgtttcag 720 caaatgcgag atgtacttggaacatttgat actgctcaaa taataaaact cctccctttt 780 gccgctgctc ctccggaacagagtaggatg cagttctctt ctttgactgt taatgtaaga 840 ggatcgggaa tgaggatacttgtaagaggc aattccccag tgttcaacta caataaagcc 900 actaagaggc tcacagtcctcggaaaggat gcaggtgcgc ttactgaaga cccagatgaa 960 ggtacggctg gagtagaatctgctgttcta agagggtttc tcattttagg taaagaaaac 1020 aagagatatg gcccagcactaagcatcaat gaactgagca aacttgcaaa aggggagaaa 1080 gctaatgtgc taattgggcaaggggacgtg gtgttggtaa tgaaacggaa acgtgactct 1140 agcatactta ctgacagccagacagcgacc aaaaggattc ggatggccat caat 1194 22 1232 DNA Equine influenzavirus H3N8 22 agaattcaca atggtcggaa gaagagcaac agccattctc agaaaggcaaccagaagatt 60 gattcaattg atagtaagtg ggagagatga acaatcaatt gctgaagcaataattgtagc 120 catggtgttt tcgcaagaag attgcatgat aaaagcagtt cgaggcgatttgaacttcgt 180 taatagagca aatcagcgct tgaaccccat gcatcaactc ttgaggcatttccaaaaaga 240 tgcaaaagtg cttttccaga attgggggat tgaacccatc gacaatgtgatgggaatgat 300 tggaatattg cctgacatga ccccaagcac cgagatgtca ttgagaggagtgagagtcag 360 caaaatggga gtggatgagt actccagcac tgagagagtg gtggtgagcattgaccgttt 420 tttaagagtt cgggatcaaa ggggaaacat actactgtcc cctgaagaggtcagtgaaac 480 acaaggaacg gaaaagctga caataattta ttcatcatca atgatgtgggagattaatgg 540 tcccgaatca gtgttggtca atacttatca atggatcatc aggaactgggaaattgtgaa 600 aattcaatgg tcacaggatc ccacaatgtt atacaataag atagaatttgagccattcca 660 gtccctggtc cctagggcca ccagaagcca atacagcggt ttcgtaagaaccctgtttca 720 gcaaatgcga gatgtacttg gaacatttga tactgctcaa ataataaaactcctcccttt 780 tgccgctgct cctccggaac agagtaggat gcagttctct tctttgactgttaatgtaag 840 aggatcggga atgaggatac ttgtaagagg caattcccca gtgttcaactacaataaagc 900 cactaagagg ctcacagtcc tcggaaagga tgcaggtgcg cttactgaagacccagatga 960 aggtacggct ggagtagaat ctgctgttct aagagggttt ctcattttaggtaaagaaaa 1020 caagagatat ggcccagcac taagcatcaa tgaactgagc aaacttgcaaaaggggagaa 1080 agctaatgtg ctaattgggc aaggggacgt ggtgttggta atgaaacggaaacgtgactc 1140 tagcatactt actgacagcc agacagcgac caaaaggatt cggatggccatcaattagtg 1200 ttgaattgtt taaaaacgac cttgtttcta ct 1232 23 1232 DNAEquine influenza virus H3N8 CDS (2)..(1195) 23 a gaa ttc aca atg gtc ggaaga aga gca aca gcc att ctc aga aag gca 49 Glu Phe Thr Met Val Gly ArgArg Ala Thr Ala Ile Leu Arg Lys Ala 1 5 10 15 acc aga aga ttg att caattg ata gta agt ggg aga gat gaa caa tca 97 Thr Arg Arg Leu Ile Gln LeuIle Val Ser Gly Arg Asp Glu Gln Ser 20 25 30 att gct gaa gca ata att gtagcc atg gtg ttt tcg caa gaa gat tgc 145 Ile Ala Glu Ala Ile Ile Val AlaMet Val Phe Ser Gln Glu Asp Cys 35 40 45 atg ata caa gca gtt cga ggc gatttg aac ttc gtt aat aga gca aat 193 Met Ile Gln Ala Val Arg Gly Asp LeuAsn Phe Val Asn Arg Ala Asn 50 55 60 cag cgc ttg aac ccc atg cat caa ctcttg agg cat ttc caa aaa gat 241 Gln Arg Leu Asn Pro Met His Gln Leu LeuArg His Phe Gln Lys Asp 65 70 75 80 gca aaa gtg ctt ttc cag aat tgg gggatt gaa ccc atc gac aat gtg 289 Ala Lys Val Leu Phe Gln Asn Trp Gly IleGlu Pro Ile Asp Asn Val 85 90 95 atg gga atg att gga ata ttg cct gac atgacc cca agc acc gag atg 337 Met Gly Met Ile Gly Ile Leu Pro Asp Met ThrPro Ser Thr Glu Met 100 105 110 tca ttg aga gga gtg aga gtc agc aaa atggga gtg gat gag tac tcc 385 Ser Leu Arg Gly Val Arg Val Ser Lys Met GlyVal Asp Glu Tyr Ser 115 120 125 agc act gag aga gtg gtg gtg agc att gaccgt ttt tta aga gtt cgg 433 Ser Thr Glu Arg Val Val Val Ser Ile Asp ArgPhe Leu Arg Val Arg 130 135 140 gat caa agg gga aac ata cta ctg tcc cctgaa gag gtc agt gaa aca 481 Asp Gln Arg Gly Asn Ile Leu Leu Ser Pro GluGlu Val Ser Glu Thr 145 150 155 160 caa gga acg gaa aag ctg aca ata atttat tca tca tca atg atg tgg 529 Gln Gly Thr Glu Lys Leu Thr Ile Ile TyrSer Ser Ser Met Met Trp 165 170 175 gag att aat ggt ccc gaa tca gtg ttggtc aat act tat caa tgg atc 577 Glu Ile Asn Gly Pro Glu Ser Val Leu ValAsn Thr Tyr Gln Trp Ile 180 185 190 atc agg aac tgg gaa att gtg aaa attcaa tgg tca cag gat ccc aca 625 Ile Arg Asn Trp Glu Ile Val Lys Ile GlnTrp Ser Gln Asp Pro Thr 195 200 205 atg tta tac aat aag ata gaa ttt gagcca ttc cag tcc ctg gtc cct 673 Met Leu Tyr Asn Lys Ile Glu Phe Glu ProPhe Gln Ser Leu Val Pro 210 215 220 agg gcc acc aga agc caa tac agc ggtttc gta aga acc ctg ttt cag 721 Arg Ala Thr Arg Ser Gln Tyr Ser Gly PheVal Arg Thr Leu Phe Gln 225 230 235 240 caa atg cga gat gta ctt gga acattt gat act gct caa ata ata aaa 769 Gln Met Arg Asp Val Leu Gly Thr PheAsp Thr Ala Gln Ile Ile Lys 245 250 255 ctc ctc cct ttt gcc gct gct cctccg gaa cag agt agg atg cag ttc 817 Leu Leu Pro Phe Ala Ala Ala Pro ProGlu Gln Ser Arg Met Gln Phe 260 265 270 tct tct ttg act gtt aat gta agagga tcg gga atg agg ata ctt gta 865 Ser Ser Leu Thr Val Asn Val Arg GlySer Gly Met Arg Ile Leu Val 275 280 285 aga ggc aat tcc cca gtg ttc aactac aat aaa gcc act aag agg ctc 913 Arg Gly Asn Ser Pro Val Phe Asn TyrAsn Lys Ala Thr Lys Arg Leu 290 295 300 aca gtc ctc gga aaa gat gca ggtgcg ctt act gaa gac cca gat gaa 961 Thr Val Leu Gly Lys Asp Ala Gly AlaLeu Thr Glu Asp Pro Asp Glu 305 310 315 320 ggt acg gct gga gta gaa tctgct gtt cta aga ggg ttt ctc att tta 1009 Gly Thr Ala Gly Val Glu Ser AlaVal Leu Arg Gly Phe Leu Ile Leu 325 330 335 ggt aaa gaa aac aag aga tatggc cca gca cta agc atc aat gaa ctg 1057 Gly Lys Glu Asn Lys Arg Tyr GlyPro Ala Leu Ser Ile Asn Glu Leu 340 345 350 agc aaa ctt gca aaa ggg gagaaa gct aat gtg cta att ggg caa ggg 1105 Ser Lys Leu Ala Lys Gly Glu LysAla Asn Val Leu Ile Gly Gln Gly 355 360 365 gac gtg gtg ttg gta atg aaacgg aaa cgt gac tct agc ata ctt act 1153 Asp Val Val Leu Val Met Lys ArgLys Arg Asp Ser Ser Ile Leu Thr 370 375 380 gac agc cag aca gcg acc aaaagg att cgg atg gcc atc aat 1195 Asp Ser Gln Thr Ala Thr Lys Arg Ile ArgMet Ala Ile Asn 385 390 395 tagtgttgaa ttgtttaaaa acgaccttgt ttctact1232 24 398 PRT Equine influenza virus H3N8 24 Glu Phe Thr Met Val GlyArg Arg Ala Thr Ala Ile Leu Arg Lys Ala 1 5 10 15 Thr Arg Arg Leu IleGln Leu Ile Val Ser Gly Arg Asp Glu Gln Ser 20 25 30 Ile Ala Glu Ala IleIle Val Ala Met Val Phe Ser Gln Glu Asp Cys 35 40 45 Met Ile Gln Ala ValArg Gly Asp Leu Asn Phe Val Asn Arg Ala Asn 50 55 60 Gln Arg Leu Asn ProMet His Gln Leu Leu Arg His Phe Gln Lys Asp 65 70 75 80 Ala Lys Val LeuPhe Gln Asn Trp Gly Ile Glu Pro Ile Asp Asn Val 85 90 95 Met Gly Met IleGly Ile Leu Pro Asp Met Thr Pro Ser Thr Glu Met 100 105 110 Ser Leu ArgGly Val Arg Val Ser Lys Met Gly Val Asp Glu Tyr Ser 115 120 125 Ser ThrGlu Arg Val Val Val Ser Ile Asp Arg Phe Leu Arg Val Arg 130 135 140 AspGln Arg Gly Asn Ile Leu Leu Ser Pro Glu Glu Val Ser Glu Thr 145 150 155160 Gln Gly Thr Glu Lys Leu Thr Ile Ile Tyr Ser Ser Ser Met Met Trp 165170 175 Glu Ile Asn Gly Pro Glu Ser Val Leu Val Asn Thr Tyr Gln Trp Ile180 185 190 Ile Arg Asn Trp Glu Ile Val Lys Ile Gln Trp Ser Gln Asp ProThr 195 200 205 Met Leu Tyr Asn Lys Ile Glu Phe Glu Pro Phe Gln Ser LeuVal Pro 210 215 220 Arg Ala Thr Arg Ser Gln Tyr Ser Gly Phe Val Arg ThrLeu Phe Gln 225 230 235 240 Gln Met Arg Asp Val Leu Gly Thr Phe Asp ThrAla Gln Ile Ile Lys 245 250 255 Leu Leu Pro Phe Ala Ala Ala Pro Pro GluGln Ser Arg Met Gln Phe 260 265 270 Ser Ser Leu Thr Val Asn Val Arg GlySer Gly Met Arg Ile Leu Val 275 280 285 Arg Gly Asn Ser Pro Val Phe AsnTyr Asn Lys Ala Thr Lys Arg Leu 290 295 300 Thr Val Leu Gly Lys Asp AlaGly Ala Leu Thr Glu Asp Pro Asp Glu 305 310 315 320 Gly Thr Ala Gly ValGlu Ser Ala Val Leu Arg Gly Phe Leu Ile Leu 325 330 335 Gly Lys Glu AsnLys Arg Tyr Gly Pro Ala Leu Ser Ile Asn Glu Leu 340 345 350 Ser Lys LeuAla Lys Gly Glu Lys Ala Asn Val Leu Ile Gly Gln Gly 355 360 365 Asp ValVal Leu Val Met Lys Arg Lys Arg Asp Ser Ser Ile Leu Thr 370 375 380 AspSer Gln Thr Ala Thr Lys Arg Ile Arg Met Ala Ile Asn 385 390 395 25 1194DNA Equine influenza virus H3N8 25 gaattcacaa tggtcggaag aagagcaacagccattctca gaaaggcaac cagaagattg 60 attcaattga tagtaagtgg gagagatgaacaatcaattg ctgaagcaat aattgtagcc 120 atggtgtttt cgcaagaaga ttgcatgatacaagcagttc gaggcgattt gaacttcgtt 180 aatagagcaa atcagcgctt gaaccccatgcatcaactct tgaggcattt ccaaaaagat 240 gcaaaagtgc ttttccagaa ttgggggattgaacccatcg acaatgtgat gggaatgatt 300 ggaatattgc ctgacatgac cccaagcaccgagatgtcat tgagaggagt gagagtcagc 360 aaaatgggag tggatgagta ctccagcactgagagagtgg tggtgagcat tgaccgtttt 420 ttaagagttc gggatcaaag gggaaacatactactgtccc ctgaagaggt cagtgaaaca 480 caaggaacgg aaaagctgac aataatttattcatcatcaa tgatgtggga gattaatggt 540 cccgaatcag tgttggtcaa tacttatcaatggatcatca ggaactggga aattgtgaaa 600 attcaatggt cacaggatcc cacaatgttatacaataaga tagaatttga gccattccag 660 tccctggtcc ctagggccac cagaagccaatacagcggtt tcgtaagaac cctgtttcag 720 caaatgcgag atgtacttgg aacatttgatactgctcaaa taataaaact cctccctttt 780 gccgctgctc ctccggaaca gagtaggatgcagttctctt ctttgactgt taatgtaaga 840 ggatcgggaa tgaggatact tgtaagaggcaattccccag tgttcaacta caataaagcc 900 actaagaggc tcacagtcct cggaaaagatgcaggtgcgc ttactgaaga cccagatgaa 960 ggtacggctg gagtagaatc tgctgttctaagagggtttc tcattttagg taaagaaaac 1020 aagagatatg gcccagcact aagcatcaatgaactgagca aacttgcaaa aggggagaaa 1080 gctaatgtgc taattgggca aggggacgtggtgttggtaa tgaaacggaa acgtgactct 1140 agcatactta ctgacagcca gacagcgaccaaaaggattc ggatggccat caat 1194 26 23 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Primer 26 agcaaaagcaggtagatatt gaa 23 27 24 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Primer 27 agtagaaaca aggtagtttt ttac 24 2818 DNA Artificial Sequence Description of Artificial Sequence SyntheticPrimer 28 caggaaacag ctatgacc 18 29 20 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Primer 29 taatacgactcactataggg 20 30 18 DNA Artificial Sequence Description of ArtificialSequence Synthetic Primer 30 tggtgcacta gccagctg 18 31 18 DNA ArtificialSequence Description of Artificial Sequence Synthetic Primer 31ttgcctgtac catctgcc 18 32 23 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Primer 32 agcaaaagca ggggatattt ctg 23 3323 DNA Artificial Sequence Description of Artificial Sequence SyntheticPrimer 33 agtagaaaca agggtgtttt taa 23 34 16 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Primer 34 gacatccctg actatg16 35 16 DNA Artificial Sequence Description of Artificial SequenceSynthetic Primer 35 gcatctgtta agtcaa 16 36 25 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Primer 36 agcaaaagcaggtcaaatat attca 25 37 26 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Primer 37 gaaaacacca tggctacaat tattgc 2638 27 DNA Artificial Sequence Description of Artificial SequenceSynthetic Primer 38 agaattcaca atggtcggaa gaagagc 27 39 27 DNAArtificial Sequence Description of Artificial Sequence Synthetic Primer39 agtagaaaca aggtcgtttt taaacaa 27 40 19 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Primer 40 agccgtaccttcatctggg 19 41 19 DNA Artificial Sequence Description of ArtificialSequence Synthetic Primer 41 agcactgaga gagtggtgg 19 42 19 DNAArtificial Sequence Description of Artificial Sequence Synthetic Primer42 gtaagaggca attccccag 19 43 18 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Primer 43 cagcttttcc gttccttg 18 44 2341DNA Equine influenza virus H3N8 CDS (28)..(2304) 44 agcaaaagcaggtcaaatat attcaat atg gag aga ata aaa gaa ctg aga gat 54 Met Glu ArgIle Lys Glu Leu Arg Asp 1 5 cta atg tca caa tcc cgc acc cgc gag ata ctaaca aaa act act gtg 102 Leu Met Ser Gln Ser Arg Thr Arg Glu Ile Leu ThrLys Thr Thr Val 10 15 20 25 gac cac atg gcc ata atc aag aaa tac aca tcagga aga caa gag aag 150 Asp His Met Ala Ile Ile Lys Lys Tyr Thr Ser GlyArg Gln Glu Lys 30 35 40 aac ccc gca ctt agg atg aag tgg atg atg gca atgaaa tac cca att 198 Asn Pro Ala Leu Arg Met Lys Trp Met Met Ala Met LysTyr Pro Ile 45 50 55 aca gca gat aag agg ata atg gaa atg att cct gag agaaat gaa cag 246 Thr Ala Asp Lys Arg Ile Met Glu Met Ile Pro Glu Arg AsnGlu Gln 60 65 70 ggg caa acc ctt tgg agc aaa acg aac gat gct ggc tca gaccgc gta 294 Gly Gln Thr Leu Trp Ser Lys Thr Asn Asp Ala Gly Ser Asp ArgVal 75 80 85 atg gta tca cct ctg gca gtg aca tgg tgg aat agg aat gga ccaaca 342 Met Val Ser Pro Leu Ala Val Thr Trp Trp Asn Arg Asn Gly Pro Thr90 95 100 105 acg agc aca att cat tat cca aaa gtc tac aaa act tat tttgaa aaa 390 Thr Ser Thr Ile His Tyr Pro Lys Val Tyr Lys Thr Tyr Phe GluLys 110 115 120 gtt gaa aga tta aaa cac gga acc ttt ggc ccc gtt cat tttagg aat 438 Val Glu Arg Leu Lys His Gly Thr Phe Gly Pro Val His Phe ArgAsn 125 130 135 caa gtc aag ata aga cgg aga gtt gat gta aac cct ggt cacgcg gac 486 Gln Val Lys Ile Arg Arg Arg Val Asp Val Asn Pro Gly His AlaAsp 140 145 150 ctc agt gcc aaa gaa gca caa gat gtg atc atg gaa gtt gttttc cca 534 Leu Ser Ala Lys Glu Ala Gln Asp Val Ile Met Glu Val Val PhePro 155 160 165 aat gaa gtg gga gcc aga att cta aca tcg gaa tca caa ctaaca ata 582 Asn Glu Val Gly Ala Arg Ile Leu Thr Ser Glu Ser Gln Leu ThrIle 170 175 180 185 acc aaa gag aaa aaa gaa gaa ctt cag gac tgc aaa attgcc ccc ttg 630 Thr Lys Glu Lys Lys Glu Glu Leu Gln Asp Cys Lys Ile AlaPro Leu 190 195 200 atg gta gca tac atg cta gaa aga gag ttg gtc cga aaaaca aga ttc 678 Met Val Ala Tyr Met Leu Glu Arg Glu Leu Val Arg Lys ThrArg Phe 205 210 215 ctc cca gtg gct ggc gga aca agc agt gta tac att gaagtg ttg cat 726 Leu Pro Val Ala Gly Gly Thr Ser Ser Val Tyr Ile Glu ValLeu His 220 225 230 ctg act cag gga aca tgc tgg gaa caa atg tac acc ccagga gga gaa 774 Leu Thr Gln Gly Thr Cys Trp Glu Gln Met Tyr Thr Pro GlyGly Glu 235 240 245 gtt aga aac gat gac att gat caa agt tta att att gctgcc cgg aac 822 Val Arg Asn Asp Asp Ile Asp Gln Ser Leu Ile Ile Ala AlaArg Asn 250 255 260 265 ata gtg aga aga gcg aca gta tca gca gat cca ctagca tcc ctg ctg 870 Ile Val Arg Arg Ala Thr Val Ser Ala Asp Pro Leu AlaSer Leu Leu 270 275 280 gaa atg tgc cac agt aca cag att ggt gga ata aggatg gta gac atc 918 Glu Met Cys His Ser Thr Gln Ile Gly Gly Ile Arg MetVal Asp Ile 285 290 295 ctt aag cag aat cca aca gag gaa caa gct gtg gatata tgc aaa gca 966 Leu Lys Gln Asn Pro Thr Glu Glu Gln Ala Val Asp IleCys Lys Ala 300 305 310 gca atg ggg tta aga att agc tca tca ttc agc tttggt gga ttc acc 1014 Ala Met Gly Leu Arg Ile Ser Ser Ser Phe Ser Phe GlyGly Phe Thr 315 320 325 ttt aag aga aca agt gga tca tca gtc aag aga gaagaa gaa atg ctt 1062 Phe Lys Arg Thr Ser Gly Ser Ser Val Lys Arg Glu GluGlu Met Leu 330 335 340 345 acg ggc aac ctt caa aca ttg aaa ata aga gtgcat gaa ggc tat gaa 1110 Thr Gly Asn Leu Gln Thr Leu Lys Ile Arg Val HisGlu Gly Tyr Glu 350 355 360 gaa ttc aca atg gtc gga aga aga gca aca gccatt ctc aga aag gca 1158 Glu Phe Thr Met Val Gly Arg Arg Ala Thr Ala IleLeu Arg Lys Ala 365 370 375 acc aga aga ttg att caa ttg ata gta agt gggaga gat gaa caa tca 1206 Thr Arg Arg Leu Ile Gln Leu Ile Val Ser Gly ArgAsp Glu Gln Ser 380 385 390 att gct gaa gca ata att gta gcc atg gtg ttttcg caa gaa gat tgc 1254 Ile Ala Glu Ala Ile Ile Val Ala Met Val Phe SerGln Glu Asp Cys 395 400 405 atg ata aaa gca gtt cga ggc gat ttg aac ttcgtt aat aga gca aat 1302 Met Ile Lys Ala Val Arg Gly Asp Leu Asn Phe ValAsn Arg Ala Asn 410 415 420 425 cag cgc ttg aac ccc atg cat caa ctc ttgagg cat ttc caa aaa gat 1350 Gln Arg Leu Asn Pro Met His Gln Leu Leu ArgHis Phe Gln Lys Asp 430 435 440 gca aaa gtg ctt ttc cag aat tgg ggg attgaa ccc atc gac aat gtg 1398 Ala Lys Val Leu Phe Gln Asn Trp Gly Ile GluPro Ile Asp Asn Val 445 450 455 atg gga atg att gga ata ttg cct gac atgacc cca agc acc gag atg 1446 Met Gly Met Ile Gly Ile Leu Pro Asp Met ThrPro Ser Thr Glu Met 460 465 470 tca ttg aga gga gtg aga gtc agc aaa atggga gtg gat gag tac tcc 1494 Ser Leu Arg Gly Val Arg Val Ser Lys Met GlyVal Asp Glu Tyr Ser 475 480 485 agc act gag aga gtg gtg gtg agc att gaccgt ttt tta aga gtt cgg 1542 Ser Thr Glu Arg Val Val Val Ser Ile Asp ArgPhe Leu Arg Val Arg 490 495 500 505 gat caa agg gga aac ata cta ctg tcccct gaa gag gtc agt gaa aca 1590 Asp Gln Arg Gly Asn Ile Leu Leu Ser ProGlu Glu Val Ser Glu Thr 510 515 520 caa gga acg gaa aag ctg aca ata atttat tca tca tca atg atg tgg 1638 Gln Gly Thr Glu Lys Leu Thr Ile Ile TyrSer Ser Ser Met Met Trp 525 530 535 gag att aat ggt ccc gaa tca gtg ttggtc aat act tat caa tgg atc 1686 Glu Ile Asn Gly Pro Glu Ser Val Leu ValAsn Thr Tyr Gln Trp Ile 540 545 550 atc agg aac tgg gaa att gtg aaa attcaa tgg tca cag gat ccc aca 1734 Ile Arg Asn Trp Glu Ile Val Lys Ile GlnTrp Ser Gln Asp Pro Thr 555 560 565 atg tta tac aat aag ata gaa ttt gagcca ttc cag tcc ctg gtc cct 1782 Met Leu Tyr Asn Lys Ile Glu Phe Glu ProPhe Gln Ser Leu Val Pro 570 575 580 585 agg gcc acc aga agc caa tac agcggt ttc gta aga acc ctg ttt cag 1830 Arg Ala Thr Arg Ser Gln Tyr Ser GlyPhe Val Arg Thr Leu Phe Gln 590 595 600 caa atg cga gat gta ctt gga acattt gat act gct caa ata ata aaa 1878 Gln Met Arg Asp Val Leu Gly Thr PheAsp Thr Ala Gln Ile Ile Lys 605 610 615 ctc ctc cct ttt gcc gct gct cctccg gaa cag agt agg atg cag ttc 1926 Leu Leu Pro Phe Ala Ala Ala Pro ProGlu Gln Ser Arg Met Gln Phe 620 625 630 tct tct ttg act gtt aat gta agagga tcg gga atg agg ata ctt gta 1974 Ser Ser Leu Thr Val Asn Val Arg GlySer Gly Met Arg Ile Leu Val 635 640 645 aga ggc aat tcc cca gtg ttc aactac aat aaa gcc act aag agg ctc 2022 Arg Gly Asn Ser Pro Val Phe Asn TyrAsn Lys Ala Thr Lys Arg Leu 650 655 660 665 aca gtc ctc gga aag gat gcaggt gcg ctt act gaa gac cca gat gaa 2070 Thr Val Leu Gly Lys Asp Ala GlyAla Leu Thr Glu Asp Pro Asp Glu 670 675 680 ggt acg gct gga gta gaa tctgct gtt cta aga ggg ttt ctc att tta 2118 Gly Thr Ala Gly Val Glu Ser AlaVal Leu Arg Gly Phe Leu Ile Leu 685 690 695 ggt aaa gaa aac aag aga tatggc cca gca cta agc atc aat gaa ctg 2166 Gly Lys Glu Asn Lys Arg Tyr GlyPro Ala Leu Ser Ile Asn Glu Leu 700 705 710 agc aaa ctt gca aaa ggg gagaaa gct aat gtg cta att ggg caa ggg 2214 Ser Lys Leu Ala Lys Gly Glu LysAla Asn Val Leu Ile Gly Gln Gly 715 720 725 gac gtg gtg ttg gta atg aaacgg aaa cgt gac tct agc ata ctt act 2262 Asp Val Val Leu Val Met Lys ArgLys Arg Asp Ser Ser Ile Leu Thr 730 735 740 745 gac agc cag aca gcg accaaa agg att cgg atg gcc atc aat 2304 Asp Ser Gln Thr Ala Thr Lys Arg IleArg Met Ala Ile Asn 750 755 tagtgttgaa ttgtttaaaa acgaccttgt ttctact2341 45 759 PRT Equine influenza virus H3N8 45 Met Glu Arg Ile Lys GluLeu Arg Asp Leu Met Ser Gln Ser Arg Thr 1 5 10 15 Arg Glu Ile Leu ThrLys Thr Thr Val Asp His Met Ala Ile Ile Lys 20 25 30 Lys Tyr Thr Ser GlyArg Gln Glu Lys Asn Pro Ala Leu Arg Met Lys 35 40 45 Trp Met Met Ala MetLys Tyr Pro Ile Thr Ala Asp Lys Arg Ile Met 50 55 60 Glu Met Ile Pro GluArg Asn Glu Gln Gly Gln Thr Leu Trp Ser Lys 65 70 75 80 Thr Asn Asp AlaGly Ser Asp Arg Val Met Val Ser Pro Leu Ala Val 85 90 95 Thr Trp Trp AsnArg Asn Gly Pro Thr Thr Ser Thr Ile His Tyr Pro 100 105 110 Lys Val TyrLys Thr Tyr Phe Glu Lys Val Glu Arg Leu Lys His Gly 115 120 125 Thr PheGly Pro Val His Phe Arg Asn Gln Val Lys Ile Arg Arg Arg 130 135 140 ValAsp Val Asn Pro Gly His Ala Asp Leu Ser Ala Lys Glu Ala Gln 145 150 155160 Asp Val Ile Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg Ile 165170 175 Leu Thr Ser Glu Ser Gln Leu Thr Ile Thr Lys Glu Lys Lys Glu Glu180 185 190 Leu Gln Asp Cys Lys Ile Ala Pro Leu Met Val Ala Tyr Met LeuGlu 195 200 205 Arg Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Ala GlyGly Thr 210 215 220 Ser Ser Val Tyr Ile Glu Val Leu His Leu Thr Gln GlyThr Cys Trp 225 230 235 240 Glu Gln Met Tyr Thr Pro Gly Gly Glu Val ArgAsn Asp Asp Ile Asp 245 250 255 Gln Ser Leu Ile Ile Ala Ala Arg Asn IleVal Arg Arg Ala Thr Val 260 265 270 Ser Ala Asp Pro Leu Ala Ser Leu LeuGlu Met Cys His Ser Thr Gln 275 280 285 Ile Gly Gly Ile Arg Met Val AspIle Leu Lys Gln Asn Pro Thr Glu 290 295 300 Glu Gln Ala Val Asp Ile CysLys Ala Ala Met Gly Leu Arg Ile Ser 305 310 315 320 Ser Ser Phe Ser PheGly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser 325 330 335 Ser Val Lys ArgGlu Glu Glu Met Leu Thr Gly Asn Leu Gln Thr Leu 340 345 350 Lys Ile ArgVal His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Arg 355 360 365 Arg AlaThr Ala Ile Leu Arg Lys Ala Thr Arg Arg Leu Ile Gln Leu 370 375 380 IleVal Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val 385 390 395400 Ala Met Val Phe Ser Gln Glu Asp Cys Met Ile Lys Ala Val Arg Gly 405410 415 Asp Leu Asn Phe Val Asn Arg Ala Asn Gln Arg Leu Asn Pro Met His420 425 430 Gln Leu Leu Arg His Phe Gln Lys Asp Ala Lys Val Leu Phe GlnAsn 435 440 445 Trp Gly Ile Glu Pro Ile Asp Asn Val Met Gly Met Ile GlyIle Leu 450 455 460 Pro Asp Met Thr Pro Ser Thr Glu Met Ser Leu Arg GlyVal Arg Val 465 470 475 480 Ser Lys Met Gly Val Asp Glu Tyr Ser Ser ThrGlu Arg Val Val Val 485 490 495 Ser Ile Asp Arg Phe Leu Arg Val Arg AspGln Arg Gly Asn Ile Leu 500 505 510 Leu Ser Pro Glu Glu Val Ser Glu ThrGln Gly Thr Glu Lys Leu Thr 515 520 525 Ile Ile Tyr Ser Ser Ser Met MetTrp Glu Ile Asn Gly Pro Glu Ser 530 535 540 Val Leu Val Asn Thr Tyr GlnTrp Ile Ile Arg Asn Trp Glu Ile Val 545 550 555 560 Lys Ile Gln Trp SerGln Asp Pro Thr Met Leu Tyr Asn Lys Ile Glu 565 570 575 Phe Glu Pro PheGln Ser Leu Val Pro Arg Ala Thr Arg Ser Gln Tyr 580 585 590 Ser Gly PheVal Arg Thr Leu Phe Gln Gln Met Arg Asp Val Leu Gly 595 600 605 Thr PheAsp Thr Ala Gln Ile Ile Lys Leu Leu Pro Phe Ala Ala Ala 610 615 620 ProPro Glu Gln Ser Arg Met Gln Phe Ser Ser Leu Thr Val Asn Val 625 630 635640 Arg Gly Ser Gly Met Arg Ile Leu Val Arg Gly Asn Ser Pro Val Phe 645650 655 Asn Tyr Asn Lys Ala Thr Lys Arg Leu Thr Val Leu Gly Lys Asp Ala660 665 670 Gly Ala Leu Thr Glu Asp Pro Asp Glu Gly Thr Ala Gly Val GluSer 675 680 685 Ala Val Leu Arg Gly Phe Leu Ile Leu Gly Lys Glu Asn LysArg Tyr 690 695 700 Gly Pro Ala Leu Ser Ile Asn Glu Leu Ser Lys Leu AlaLys Gly Glu 705 710 715 720 Lys Ala Asn Val Leu Ile Gly Gln Gly Asp ValVal Leu Val Met Lys 725 730 735 Arg Lys Arg Asp Ser Ser Ile Leu Thr AspSer Gln Thr Ala Thr Lys 740 745 750 Arg Ile Arg Met Ala Ile Asn 755 462277 DNA Equine influenza virus H3N8 46 atggagagaa taaaagaact gagagatctaatgtcacaat cccgcacccg cgagatacta 60 acaaaaacta ctgtggacca catggccataatcaagaaat acacatcagg aagacaagag 120 aagaaccccg cacttaggat gaagtggatgatggcaatga aatacccaat tacagcagat 180 aagaggataa tggaaatgat tcctgagagaaatgaacagg ggcaaaccct ttggagcaaa 240 acgaacgatg ctggctcaga ccgcgtaatggtatcacctc tggcagtgac atggtggaat 300 aggaatggac caacaacgag cacaattcattatccaaaag tctacaaaac ttattttgaa 360 aaagttgaaa gattaaaaca cggaacctttggccccgttc attttaggaa tcaagtcaag 420 ataagacgga gagttgatgt aaaccctggtcacgcggacc tcagtgccaa agaagcacaa 480 gatgtgatca tggaagttgt tttcccaaatgaagtgggag ccagaattct aacatcggaa 540 tcacaactaa caataaccaa agagaaaaaagaagaacttc aggactgcaa aattgccccc 600 ttgatggtag catacatgct agaaagagagttggtccgaa aaacaagatt cctcccagtg 660 gctggcggaa caagcagtgt atacattgaagtgttgcatc tgactcaggg aacatgctgg 720 gaacaaatgt acaccccagg aggagaagttagaaacgatg acattgatca aagtttaatt 780 attgctgccc ggaacatagt gagaagagcgacagtatcag cagatccact agcatccctg 840 ctggaaatgt gccacagtac acagattggtggaataagga tggtagacat ccttaagcag 900 aatccaacag aggaacaagc tgtggatatatgcaaagcag caatggggtt aagaattagc 960 tcatcattca gctttggtgg attcacctttaagagaacaa gtggatcatc agtcaagaga 1020 gaagaagaaa tgcttacggg caaccttcaaacattgaaaa taagagtgca tgaaggctat 1080 gaagaattca caatggtcgg aagaagagcaacagccattc tcagaaaggc aaccagaaga 1140 ttgattcaat tgatagtaag tgggagagatgaacaatcaa ttgctgaagc aataattgta 1200 gccatggtgt tttcgcaaga agattgcatgataaaagcag ttcgaggcga tttgaacttc 1260 gttaatagag caaatcagcg cttgaaccccatgcatcaac tcttgaggca tttccaaaaa 1320 gatgcaaaag tgcttttcca gaattgggggattgaaccca tcgacaatgt gatgggaatg 1380 attggaatat tgcctgacat gaccccaagcaccgagatgt cattgagagg agtgagagtc 1440 agcaaaatgg gagtggatga gtactccagcactgagagag tggtggtgag cattgaccgt 1500 tttttaagag ttcgggatca aaggggaaacatactactgt cccctgaaga ggtcagtgaa 1560 acacaaggaa cggaaaagct gacaataatttattcatcat caatgatgtg ggagattaat 1620 ggtcccgaat cagtgttggt caatacttatcaatggatca tcaggaactg ggaaattgtg 1680 aaaattcaat ggtcacagga tcccacaatgttatacaata agatagaatt tgagccattc 1740 cagtccctgg tccctagggc caccagaagccaatacagcg gtttcgtaag aaccctgttt 1800 cagcaaatgc gagatgtact tggaacatttgatactgctc aaataataaa actcctccct 1860 tttgccgctg ctcctccgga acagagtaggatgcagttct cttctttgac tgttaatgta 1920 agaggatcgg gaatgaggat acttgtaagaggcaattccc cagtgttcaa ctacaataaa 1980 gccactaaga ggctcacagt cctcggaaaggatgcaggtg cgcttactga agacccagat 2040 gaaggtacgg ctggagtaga atctgctgttctaagagggt ttctcatttt aggtaaagaa 2100 aacaagagat atggcccagc actaagcatcaatgaactga gcaaacttgc aaaaggggag 2160 aaagctaatg tgctaattgg gcaaggggacgtggtgttgg taatgaaacg gaaacgtgac 2220 tctagcatac ttactgacag ccagacagcgaccaaaagga ttcggatggc catcaat 2277 47 2341 DNA Equine influenza virusH3N8 CDS (28)..(2304) 47 agcaaaagca ggtcaaatat attcaat atg gag aga ataaaa gaa ctg aga gat 54 Met Glu Arg Ile Lys Glu Leu Arg Asp 1 5 cta atgtca caa tcc cgc acc cgc gag ata cta aca aaa act act gtg 102 Leu Met SerGln Ser Arg Thr Arg Glu Ile Leu Thr Lys Thr Thr Val 10 15 20 25 gac cacatg gcc ata atc aag aaa tac aca tca gga aga caa gag aag 150 Asp His MetAla Ile Ile Lys Lys Tyr Thr Ser Gly Arg Gln Glu Lys 30 35 40 aac ccc gcactt agg atg aag tgg atg atg gca atg aaa tac cca att 198 Asn Pro Ala LeuArg Met Lys Trp Met Met Ala Met Lys Tyr Pro Ile 45 50 55 aca gca gat aagagg ata atg gaa atg att cct gag aga aat gaa cag 246 Thr Ala Asp Lys ArgIle Met Glu Met Ile Pro Glu Arg Asn Glu Gln 60 65 70 ggg caa acc ctt tggagc aaa acg aac gat gct ggc tca gac cgc gta 294 Gly Gln Thr Leu Trp SerLys Thr Asn Asp Ala Gly Ser Asp Arg Val 75 80 85 atg gta tca cct ctg gcagtg aca tgg tgg aat agg aat gga cca aca 342 Met Val Ser Pro Leu Ala ValThr Trp Trp Asn Arg Asn Gly Pro Thr 90 95 100 105 acg agc aca att cattat cca aaa gtc cac aaa act tat ttt gaa aaa 390 Thr Ser Thr Ile His TyrPro Lys Val His Lys Thr Tyr Phe Glu Lys 110 115 120 gtt gaa aga tta aaacac gga acc ttt ggc ccc gtt cat ttt agg aat 438 Val Glu Arg Leu Lys HisGly Thr Phe Gly Pro Val His Phe Arg Asn 125 130 135 caa gtc aag ata agacgg aga gtt gat gta aac cct ggt cac gcg gac 486 Gln Val Lys Ile Arg ArgArg Val Asp Val Asn Pro Gly His Ala Asp 140 145 150 ctc agt gcc aaa gaagca caa gat gtg atc atg gaa gtt gtt ttc cca 534 Leu Ser Ala Lys Glu AlaGln Asp Val Ile Met Glu Val Val Phe Pro 155 160 165 aat gaa gtg gga gccaga att cta aca tcg gaa tca caa cta aca ata 582 Asn Glu Val Gly Ala ArgIle Leu Thr Ser Glu Ser Gln Leu Thr Ile 170 175 180 185 acc aaa gag aaaaaa gaa gaa ctt cag gac tgc aaa att gcc ccc ttg 630 Thr Lys Glu Lys LysGlu Glu Leu Gln Asp Cys Lys Ile Ala Pro Leu 190 195 200 atg gta gca tacatg cta gaa aga gag ttg gtc cga aaa aca aga ttc 678 Met Val Ala Tyr MetLeu Glu Arg Glu Leu Val Arg Lys Thr Arg Phe 205 210 215 ctc cca gtg gctggc gga aca agc agt gta tac att gaa gtg ttg cat 726 Leu Pro Val Ala GlyGly Thr Ser Ser Val Tyr Ile Glu Val Leu His 220 225 230 ctg act cag ggaaca tgc tgg gaa caa atg tac acc cca gga gga gaa 774 Leu Thr Gln Gly ThrCys Trp Glu Gln Met Tyr Thr Pro Gly Gly Glu 235 240 245 gtt aga aac gatgac att gat caa agt tta att att gct gcc cgg aac 822 Val Arg Asn Asp AspIle Asp Gln Ser Leu Ile Ile Ala Ala Arg Asn 250 255 260 265 ata gtg agaaga gcg aca gta tca gca gat cca cta gca tcc ctg ctg 870 Ile Val Arg ArgAla Thr Val Ser Ala Asp Pro Leu Ala Ser Leu Leu 270 275 280 gaa atg tgccac agt aca cag att ggt gga ata agg atg gta gac atc 918 Glu Met Cys HisSer Thr Gln Ile Gly Gly Ile Arg Met Val Asp Ile 285 290 295 ctt aag cagaat cca aca gag gaa caa gct gtg gat ata tgc aaa gca 966 Leu Lys Gln AsnPro Thr Glu Glu Gln Ala Val Asp Ile Cys Lys Ala 300 305 310 gca atg gggtta aga att agc tca tca ttc agc ttt ggt gga ttc acc 1014 Ala Met Gly LeuArg Ile Ser Ser Ser Phe Ser Phe Gly Gly Phe Thr 315 320 325 ttt aag agaaca agt gga tca tca gtc aag aga gaa gaa gaa atg ctt 1062 Phe Lys Arg ThrSer Gly Ser Ser Val Lys Arg Glu Glu Glu Met Leu 330 335 340 345 acg ggcaac ctt caa aca ttg aaa ata aga gtg cat gaa ggc tat gaa 1110 Thr Gly AsnLeu Gln Thr Leu Lys Ile Arg Val His Glu Gly Tyr Glu 350 355 360 gaa ttcaca atg gtc gga aga aga gca aca gcc att ctc aga aag gca 1158 Glu Phe ThrMet Val Gly Arg Arg Ala Thr Ala Ile Leu Arg Lys Ala 365 370 375 acc agaaga ttg att caa ttg ata gta agt ggg aga gat gaa caa tca 1206 Thr Arg ArgLeu Ile Gln Leu Ile Val Ser Gly Arg Asp Glu Gln Ser 380 385 390 att gctgaa gca ata att gta gcc atg gtg ttt tcg caa gaa gat tgc 1254 Ile Ala GluAla Ile Ile Val Ala Met Val Phe Ser Gln Glu Asp Cys 395 400 405 atg atacaa gca gtt cga ggc gat ttg aac ttc gtt aat aga gca aat 1302 Met Ile GlnAla Val Arg Gly Asp Leu Asn Phe Val Asn Arg Ala Asn 410 415 420 425 cagcgc ttg aac ccc atg cat caa ctc ttg agg cat ttc caa aaa gat 1350 Gln ArgLeu Asn Pro Met His Gln Leu Leu Arg His Phe Gln Lys Asp 430 435 440 gcaaaa gtg ctt ttc cag aat tgg ggg att gaa ccc atc gac aat gtg 1398 Ala LysVal Leu Phe Gln Asn Trp Gly Ile Glu Pro Ile Asp Asn Val 445 450 455 atggga atg att gga ata ttg cct gac atg acc cca agc acc gag atg 1446 Met GlyMet Ile Gly Ile Leu Pro Asp Met Thr Pro Ser Thr Glu Met 460 465 470 tcattg aga gga gtg aga gtc agc aaa atg gga gtg gat gag tac tcc 1494 Ser LeuArg Gly Val Arg Val Ser Lys Met Gly Val Asp Glu Tyr Ser 475 480 485 agcact gag aga gtg gtg gtg agc att gac cgt ttt tta aga gtt cgg 1542 Ser ThrGlu Arg Val Val Val Ser Ile Asp Arg Phe Leu Arg Val Arg 490 495 500 505gat caa agg gga aac ata cta ctg tcc cct gaa gag gtc agt gaa aca 1590 AspGln Arg Gly Asn Ile Leu Leu Ser Pro Glu Glu Val Ser Glu Thr 510 515 520caa gga acg gaa aag ctg aca ata att tat tca tca tca atg atg tgg 1638 GlnGly Thr Glu Lys Leu Thr Ile Ile Tyr Ser Ser Ser Met Met Trp 525 530 535gag att aat ggt ccc gaa tca gtg ttg gtc aat act tat caa tgg atc 1686 GluIle Asn Gly Pro Glu Ser Val Leu Val Asn Thr Tyr Gln Trp Ile 540 545 550atc agg aac tgg gaa att gtg aaa att caa tgg tca cag gat ccc aca 1734 IleArg Asn Trp Glu Ile Val Lys Ile Gln Trp Ser Gln Asp Pro Thr 555 560 565atg tta tac aat aag ata gaa ttt gag cca ttc cag tcc ctg gtc cct 1782 MetLeu Tyr Asn Lys Ile Glu Phe Glu Pro Phe Gln Ser Leu Val Pro 570 575 580585 agg gcc acc aga agc caa tac agc ggt ttc gta aga acc ctg ttt cag 1830Arg Ala Thr Arg Ser Gln Tyr Ser Gly Phe Val Arg Thr Leu Phe Gln 590 595600 caa atg cga gat gta ctt gga aca ttt gat act gct caa ata ata aaa 1878Gln Met Arg Asp Val Leu Gly Thr Phe Asp Thr Ala Gln Ile Ile Lys 605 610615 ctc ctc cct ttt gcc gct gct cct ccg gaa cag agt agg atg cag ttc 1926Leu Leu Pro Phe Ala Ala Ala Pro Pro Glu Gln Ser Arg Met Gln Phe 620 625630 tct tct ttg act gtt aat gta aga gga tcg gga atg agg ata ctt gta 1974Ser Ser Leu Thr Val Asn Val Arg Gly Ser Gly Met Arg Ile Leu Val 635 640645 aga ggc aat tcc cca gtg ttc aac tac aat aaa gcc act aag agg ctc 2022Arg Gly Asn Ser Pro Val Phe Asn Tyr Asn Lys Ala Thr Lys Arg Leu 650 655660 665 aca gtc ctc gga aaa gat gca ggt gcg ctt act gaa gac cca gat gaa2070 Thr Val Leu Gly Lys Asp Ala Gly Ala Leu Thr Glu Asp Pro Asp Glu 670675 680 ggt acg gct gga gta gaa tct gct gtt cta aga ggg ttt ctc att tta2118 Gly Thr Ala Gly Val Glu Ser Ala Val Leu Arg Gly Phe Leu Ile Leu 685690 695 ggt aaa gaa aac aag aga tat ggc cca gca cta agc atc aat gaa ctg2166 Gly Lys Glu Asn Lys Arg Tyr Gly Pro Ala Leu Ser Ile Asn Glu Leu 700705 710 agc aaa ctt gca aaa ggg gag aaa gct aat gtg cta att ggg caa ggg2214 Ser Lys Leu Ala Lys Gly Glu Lys Ala Asn Val Leu Ile Gly Gln Gly 715720 725 gac gtg gtg ttg gta atg aaa cgg aaa cgt gac tct agc ata ctt act2262 Asp Val Val Leu Val Met Lys Arg Lys Arg Asp Ser Ser Ile Leu Thr 730735 740 745 gac agc cag aca gcg acc aaa agg att cgg atg gcc atc aat 2304Asp Ser Gln Thr Ala Thr Lys Arg Ile Arg Met Ala Ile Asn 750 755tagtgttgaa ttgtttaaaa acgaccttgt ttctact 2341 48 759 PRT Equineinfluenza virus H3N8 48 Met Glu Arg Ile Lys Glu Leu Arg Asp Leu Met SerGln Ser Arg Thr 1 5 10 15 Arg Glu Ile Leu Thr Lys Thr Thr Val Asp HisMet Ala Ile Ile Lys 20 25 30 Lys Tyr Thr Ser Gly Arg Gln Glu Lys Asn ProAla Leu Arg Met Lys 35 40 45 Trp Met Met Ala Met Lys Tyr Pro Ile Thr AlaAsp Lys Arg Ile Met 50 55 60 Glu Met Ile Pro Glu Arg Asn Glu Gln Gly GlnThr Leu Trp Ser Lys 65 70 75 80 Thr Asn Asp Ala Gly Ser Asp Arg Val MetVal Ser Pro Leu Ala Val 85 90 95 Thr Trp Trp Asn Arg Asn Gly Pro Thr ThrSer Thr Ile His Tyr Pro 100 105 110 Lys Val His Lys Thr Tyr Phe Glu LysVal Glu Arg Leu Lys His Gly 115 120 125 Thr Phe Gly Pro Val His Phe ArgAsn Gln Val Lys Ile Arg Arg Arg 130 135 140 Val Asp Val Asn Pro Gly HisAla Asp Leu Ser Ala Lys Glu Ala Gln 145 150 155 160 Asp Val Ile Met GluVal Val Phe Pro Asn Glu Val Gly Ala Arg Ile 165 170 175 Leu Thr Ser GluSer Gln Leu Thr Ile Thr Lys Glu Lys Lys Glu Glu 180 185 190 Leu Gln AspCys Lys Ile Ala Pro Leu Met Val Ala Tyr Met Leu Glu 195 200 205 Arg GluLeu Val Arg Lys Thr Arg Phe Leu Pro Val Ala Gly Gly Thr 210 215 220 SerSer Val Tyr Ile Glu Val Leu His Leu Thr Gln Gly Thr Cys Trp 225 230 235240 Glu Gln Met Tyr Thr Pro Gly Gly Glu Val Arg Asn Asp Asp Ile Asp 245250 255 Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg Ala Thr Val260 265 270 Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser ThrGln 275 280 285 Ile Gly Gly Ile Arg Met Val Asp Ile Leu Lys Gln Asn ProThr Glu 290 295 300 Glu Gln Ala Val Asp Ile Cys Lys Ala Ala Met Gly LeuArg Ile Ser 305 310 315 320 Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe LysArg Thr Ser Gly Ser 325 330 335 Ser Val Lys Arg Glu Glu Glu Met Leu ThrGly Asn Leu Gln Thr Leu 340 345 350 Lys Ile Arg Val His Glu Gly Tyr GluGlu Phe Thr Met Val Gly Arg 355 360 365 Arg Ala Thr Ala Ile Leu Arg LysAla Thr Arg Arg Leu Ile Gln Leu 370 375 380 Ile Val Ser Gly Arg Asp GluGln Ser Ile Ala Glu Ala Ile Ile Val 385 390 395 400 Ala Met Val Phe SerGln Glu Asp Cys Met Ile Gln Ala Val Arg Gly 405 410 415 Asp Leu Asn PheVal Asn Arg Ala Asn Gln Arg Leu Asn Pro Met His 420 425 430 Gln Leu LeuArg His Phe Gln Lys Asp Ala Lys Val Leu Phe Gln Asn 435 440 445 Trp GlyIle Glu Pro Ile Asp Asn Val Met Gly Met Ile Gly Ile Leu 450 455 460 ProAsp Met Thr Pro Ser Thr Glu Met Ser Leu Arg Gly Val Arg Val 465 470 475480 Ser Lys Met Gly Val Asp Glu Tyr Ser Ser Thr Glu Arg Val Val Val 485490 495 Ser Ile Asp Arg Phe Leu Arg Val Arg Asp Gln Arg Gly Asn Ile Leu500 505 510 Leu Ser Pro Glu Glu Val Ser Glu Thr Gln Gly Thr Glu Lys LeuThr 515 520 525 Ile Ile Tyr Ser Ser Ser Met Met Trp Glu Ile Asn Gly ProGlu Ser 530 535 540 Val Leu Val Asn Thr Tyr Gln Trp Ile Ile Arg Asn TrpGlu Ile Val 545 550 555 560 Lys Ile Gln Trp Ser Gln Asp Pro Thr Met LeuTyr Asn Lys Ile Glu 565 570 575 Phe Glu Pro Phe Gln Ser Leu Val Pro ArgAla Thr Arg Ser Gln Tyr 580 585 590 Ser Gly Phe Val Arg Thr Leu Phe GlnGln Met Arg Asp Val Leu Gly 595 600 605 Thr Phe Asp Thr Ala Gln Ile IleLys Leu Leu Pro Phe Ala Ala Ala 610 615 620 Pro Pro Glu Gln Ser Arg MetGln Phe Ser Ser Leu Thr Val Asn Val 625 630 635 640 Arg Gly Ser Gly MetArg Ile Leu Val Arg Gly Asn Ser Pro Val Phe 645 650 655 Asn Tyr Asn LysAla Thr Lys Arg Leu Thr Val Leu Gly Lys Asp Ala 660 665 670 Gly Ala LeuThr Glu Asp Pro Asp Glu Gly Thr Ala Gly Val Glu Ser 675 680 685 Ala ValLeu Arg Gly Phe Leu Ile Leu Gly Lys Glu Asn Lys Arg Tyr 690 695 700 GlyPro Ala Leu Ser Ile Asn Glu Leu Ser Lys Leu Ala Lys Gly Glu 705 710 715720 Lys Ala Asn Val Leu Ile Gly Gln Gly Asp Val Val Leu Val Met Lys 725730 735 Arg Lys Arg Asp Ser Ser Ile Leu Thr Asp Ser Gln Thr Ala Thr Lys740 745 750 Arg Ile Arg Met Ala Ile Asn 755 49 2277 DNA Equine influenzavirus H3N8 49 atggagagaa taaaagaact gagagatcta atgtcacaat cccgcacccgcgagatacta 60 acaaaaacta ctgtggacca catggccata atcaagaaat acacatcaggaagacaagag 120 aagaaccccg cacttaggat gaagtggatg atggcaatga aatacccaattacagcagat 180 aagaggataa tggaaatgat tcctgagaga aatgaacagg ggcaaaccctttggagcaaa 240 acgaacgatg ctggctcaga ccgcgtaatg gtatcacctc tggcagtgacatggtggaat 300 aggaatggac caacaacgag cacaattcat tatccaaaag tccacaaaacttattttgaa 360 aaagttgaaa gattaaaaca cggaaccttt ggccccgttc attttaggaatcaagtcaag 420 ataagacgga gagttgatgt aaaccctggt cacgcggacc tcagtgccaaagaagcacaa 480 gatgtgatca tggaagttgt tttcccaaat gaagtgggag ccagaattctaacatcggaa 540 tcacaactaa caataaccaa agagaaaaaa gaagaacttc aggactgcaaaattgccccc 600 ttgatggtag catacatgct agaaagagag ttggtccgaa aaacaagattcctcccagtg 660 gctggcggaa caagcagtgt atacattgaa gtgttgcatc tgactcagggaacatgctgg 720 gaacaaatgt acaccccagg aggagaagtt agaaacgatg acattgatcaaagtttaatt 780 attgctgccc ggaacatagt gagaagagcg acagtatcag cagatccactagcatccctg 840 ctggaaatgt gccacagtac acagattggt ggaataagga tggtagacatccttaagcag 900 aatccaacag aggaacaagc tgtggatata tgcaaagcag caatggggttaagaattagc 960 tcatcattca gctttggtgg attcaccttt aagagaacaa gtggatcatcagtcaagaga 1020 gaagaagaaa tgcttacggg caaccttcaa acattgaaaa taagagtgcatgaaggctat 1080 gaagaattca caatggtcgg aagaagagca acagccattc tcagaaaggcaaccagaaga 1140 ttgattcaat tgatagtaag tgggagagat gaacaatcaa ttgctgaagcaataattgta 1200 gccatggtgt tttcgcaaga agattgcatg atacaagcag ttcgaggcgatttgaacttc 1260 gttaatagag caaatcagcg cttgaacccc atgcatcaac tcttgaggcatttccaaaaa 1320 gatgcaaaag tgcttttcca gaattggggg attgaaccca tcgacaatgtgatgggaatg 1380 attggaatat tgcctgacat gaccccaagc accgagatgt cattgagaggagtgagagtc 1440 agcaaaatgg gagtggatga gtactccagc actgagagag tggtggtgagcattgaccgt 1500 tttttaagag ttcgggatca aaggggaaac atactactgt cccctgaagaggtcagtgaa 1560 acacaaggaa cggaaaagct gacaataatt tattcatcat caatgatgtgggagattaat 1620 ggtcccgaat cagtgttggt caatacttat caatggatca tcaggaactgggaaattgtg 1680 aaaattcaat ggtcacagga tcccacaatg ttatacaata agatagaatttgagccattc 1740 cagtccctgg tccctagggc caccagaagc caatacagcg gtttcgtaagaaccctgttt 1800 cagcaaatgc gagatgtact tggaacattt gatactgctc aaataataaaactcctccct 1860 tttgccgctg ctcctccgga acagagtagg atgcagttct cttctttgactgttaatgta 1920 agaggatcgg gaatgaggat acttgtaaga ggcaattccc cagtgttcaactacaataaa 1980 gccactaaga ggctcacagt cctcggaaaa gatgcaggtg cgcttactgaagacccagat 2040 gaaggtacgg ctggagtaga atctgctgtt ctaagagggt ttctcattttaggtaaagaa 2100 aacaagagat atggcccagc actaagcatc aatgaactga gcaaacttgcaaaaggggag 2160 aaagctaatg tgctaattgg gcaaggggac gtggtgttgg taatgaaacggaaacgtgac 2220 tctagcatac ttactgacag ccagacagcg accaaaagga ttcggatggccatcaat 2277 50 891 DNA Equine influenza virus H3N8 CDS (27)..(716) 50agcaaaagca gggtgacaaa aacata atg gat tcc aac act gtg tca agc ttt 53 MetAsp Ser Asn Thr Val Ser Ser Phe 1 5 cag gta gac tgt ttt ctt tgg cat gtccgc aaa cga ttt gca gac caa 101 Gln Val Asp Cys Phe Leu Trp His Val ArgLys Arg Phe Ala Asp Gln 10 15 20 25 gaa ctg ggt gat gcc cca ttc ctt gaccgg ctt cgc cga gac cag aag 149 Glu Leu Gly Asp Ala Pro Phe Leu Asp ArgLeu Arg Arg Asp Gln Lys 30 35 40 tcc cta aaa gga aga ggt agc act ctt ggtctg gac atc gaa aca gcc 197 Ser Leu Lys Gly Arg Gly Ser Thr Leu Gly LeuAsp Ile Glu Thr Ala 45 50 55 act cgt gca gga aag cag ata gtg gag cag attctg gaa gag gaa tca 245 Thr Arg Ala Gly Lys Gln Ile Val Glu Gln Ile LeuGlu Glu Glu Ser 60 65 70 gat gag gca ctt aaa atg acc att gcc tct gtt cctgct tca cgc tac 293 Asp Glu Ala Leu Lys Met Thr Ile Ala Ser Val Pro AlaSer Arg Tyr 75 80 85 tta act gac atg act ctt gat gag atg tca aga gac tggttc atg ctc 341 Leu Thr Asp Met Thr Leu Asp Glu Met Ser Arg Asp Trp PheMet Leu 90 95 100 105 atg ccc aag cag aaa gta aca ggc tcc cta tgt ataaga atg gac cag 389 Met Pro Lys Gln Lys Val Thr Gly Ser Leu Cys Ile ArgMet Asp Gln 110 115 120 gca atc atg gat aag aac atc ata ctt aaa gca aacttt agt gtg att 437 Ala Ile Met Asp Lys Asn Ile Ile Leu Lys Ala Asn PheSer Val Ile 125 130 135 ttc gaa agg ctg gag aca cta ata cta ctt aga gccttc acc gaa gaa 485 Phe Glu Arg Leu Glu Thr Leu Ile Leu Leu Arg Ala PheThr Glu Glu 140 145 150 gga gca gtc gtt ggc gaa att tca cca ttg cct tctctt cca gga cat 533 Gly Ala Val Val Gly Glu Ile Ser Pro Leu Pro Ser LeuPro Gly His 155 160 165 act aat gag gat gtc aaa aat gca att ggg gtc ctcatc gga gga ctt 581 Thr Asn Glu Asp Val Lys Asn Ala Ile Gly Val Leu IleGly Gly Leu 170 175 180 185 aaa tgg aat gat aat acg gtt aga atc tct gaaact cta cag aga ttc 629 Lys Trp Asn Asp Asn Thr Val Arg Ile Ser Glu ThrLeu Gln Arg Phe 190 195 200 gct tgg aga agc agt cat gag aat ggg aga ccttca ttc cct cca aag 677 Ala Trp Arg Ser Ser His Glu Asn Gly Arg Pro SerPhe Pro Pro Lys 205 210 215 cag aaa cga aaa atg gag aga aca att gag ccagaa gtt tgaagaaata 726 Gln Lys Arg Lys Met Glu Arg Thr Ile Glu Pro GluVal 220 225 230 agatggttga ttgaagaagt gcgacataga ttgaaaaata cagaaaatagttttgaacaa 786 ataacattta tgcaagcctt acaactattg cttgaagtag gacaagagataagaactttc 846 tcgtttcagc ttatttaatg ataaaaaaca cccttgtttc tacta 891 51230 PRT Equine influenza virus H3N8 51 Met Asp Ser Asn Thr Val Ser SerPhe Gln Val Asp Cys Phe Leu Trp 1 5 10 15 His Val Arg Lys Arg Phe AlaAsp Gln Glu Leu Gly Asp Ala Pro Phe 20 25 30 Leu Asp Arg Leu Arg Arg AspGln Lys Ser Leu Lys Gly Arg Gly Ser 35 40 45 Thr Leu Gly Leu Asp Ile GluThr Ala Thr Arg Ala Gly Lys Gln Ile 50 55 60 Val Glu Gln Ile Leu Glu GluGlu Ser Asp Glu Ala Leu Lys Met Thr 65 70 75 80 Ile Ala Ser Val Pro AlaSer Arg Tyr Leu Thr Asp Met Thr Leu Asp 85 90 95 Glu Met Ser Arg Asp TrpPhe Met Leu Met Pro Lys Gln Lys Val Thr 100 105 110 Gly Ser Leu Cys IleArg Met Asp Gln Ala Ile Met Asp Lys Asn Ile 115 120 125 Ile Leu Lys AlaAsn Phe Ser Val Ile Phe Glu Arg Leu Glu Thr Leu 130 135 140 Ile Leu LeuArg Ala Phe Thr Glu Glu Gly Ala Val Val Gly Glu Ile 145 150 155 160 SerPro Leu Pro Ser Leu Pro Gly His Thr Asn Glu Asp Val Lys Asn 165 170 175Ala Ile Gly Val Leu Ile Gly Gly Leu Lys Trp Asn Asp Asn Thr Val 180 185190 Arg Ile Ser Glu Thr Leu Gln Arg Phe Ala Trp Arg Ser Ser His Glu 195200 205 Asn Gly Arg Pro Ser Phe Pro Pro Lys Gln Lys Arg Lys Met Glu Arg210 215 220 Thr Ile Glu Pro Glu Val 225 230 52 690 DNA Equine influenzavirus H3N8 52 atggattcca acactgtgtc aagctttcag gtagactgtt ttctttggcatgtccgcaaa 60 cgatttgcag accaagaact gggtgatgcc ccattccttg accggcttcgccgagaccag 120 aagtccctaa aaggaagagg tagcactctt ggtctggaca tcgaaacagccactcgtgca 180 ggaaagcaga tagtggagca gattctggaa gaggaatcag atgaggcacttaaaatgacc 240 attgcctctg ttcctgcttc acgctactta actgacatga ctcttgatgagatgtcaaga 300 gactggttca tgctcatgcc caagcagaaa gtaacaggct ccctatgtataagaatggac 360 caggcaatca tggataagaa catcatactt aaagcaaact ttagtgtgattttcgaaagg 420 ctggagacac taatactact tagagccttc accgaagaag gagcagtcgttggcgaaatt 480 tcaccattgc cttctcttcc aggacatact aatgaggatg tcaaaaatgcaattggggtc 540 ctcatcggag gacttaaatg gaatgataat acggttagaa tctctgaaactctacagaga 600 ttcgcttgga gaagcagtca tgagaatggg agaccttcat tccctccaaagcagaaacga 660 aaaatggaga gaacaattga gccagaagtt 690 53 888 DNA Equineinfluenza virus H3N8 53 caaaagcagg gtgacaaaaa catgatggat tccaacactgtgtcaagctt tcaggtagac 60 tgttttcttt ggcatgtccg caaacgattt gcagaccaagaactgggtga tgccccattc 120 cttgaccggc ttcgccgaga ccagaagtcc ctaaaaggaagaggtagcac tcttggtctg 180 gacatcgaaa cagccactcg tgcaggaaag cagatagtggagcagattct ggaagaggaa 240 tcagatgagg cacttaaaat gaccattgcc tctgttcctgcttcacgcta cttaactgac 300 atgactcttg atgagatgtc aagagactgg ttcatgctcatgcccaagca gaaagtaaca 360 ggctccctat gtataagaat ggaccaggca atcatggataagaacatcat acttaaagca 420 aactttagtg tgattttcga aaggctggag acactaatactacttagagc cttcaccgaa 480 gaaggagcag tcgttggcga aatttcacca ttgccttctcttccaggaca tactaatgag 540 gatgtcaaaa atgcaattgg ggtcctcatc ggaggacttaaatggaatga taatacggtt 600 agaatctctg aaactctaca gagattcgct tggagaagcagtcatgagaa tgggagacct 660 tcattccctc caaagcagaa acgaaaaatg gagagaacaattgagccaga agtttgaaga 720 aataagatgg ttgattgaag aagtgcgaca tagattgaaaaatacagaaa atagttttga 780 acaaataaca tttatgcaag ccttacaact attgcttgaagtagaacaag agataagaac 840 tttctcgttt cagcttattt aatgataaaa aacacccttgtttctact 888 54 468 DNA Equine influenza virus H3N8 CDS (3)..(293) 54 acttt agt gtg att ttc gaa agg ctg gag aca cta ata cta ctt aga 47 Phe SerVal Ile Phe Glu Arg Leu Glu Thr Leu Ile Leu Leu Arg 1 5 10 15 gcc ttcacc gaa gaa gga gca gtc gtt ggc gaa att tca cca ttg cct 95 Ala Phe ThrGlu Glu Gly Ala Val Val Gly Glu Ile Ser Pro Leu Pro 20 25 30 tct ctt ccagga cat act aat gag gat gtc aaa aat gca att ggg gtc 143 Ser Leu Pro GlyHis Thr Asn Glu Asp Val Lys Asn Ala Ile Gly Val 35 40 45 ctc atc gga ggactt aaa tgg aat gat aat acg gtt aga atc tct gaa 191 Leu Ile Gly Gly LeuLys Trp Asn Asp Asn Thr Val Arg Ile Ser Glu 50 55 60 act cta cag aga ttcgct cgg aga agc agt cat gag aat ggg aga cct 239 Thr Leu Gln Arg Phe AlaArg Arg Ser Ser His Glu Asn Gly Arg Pro 65 70 75 tca ttc cct cca aag cagaaa cga aaa atg gag aga aca att gag cca 287 Ser Phe Pro Pro Lys Gln LysArg Lys Met Glu Arg Thr Ile Glu Pro 80 85 90 95 gaa gtt tgaagaaataagatggttga ttgaagaagt gcgacataga ttgaaaaata 343 Glu Val cagaaaatagttttgaacaa ataacattta tgcaagcctt acaactattg cttgaagtag 403 aacaagagataagaactttc tcgtttcagc ttatttaatg ataaaaaaca cccttgtttc 463 tacta 468 5597 PRT Equine influenza virus H3N8 55 Phe Ser Val Ile Phe Glu Arg LeuGlu Thr Leu Ile Leu Leu Arg Ala 1 5 10 15 Phe Thr Glu Glu Gly Ala ValVal Gly Glu Ile Ser Pro Leu Pro Ser 20 25 30 Leu Pro Gly His Thr Asn GluAsp Val Lys Asn Ala Ile Gly Val Leu 35 40 45 Ile Gly Gly Leu Lys Trp AsnAsp Asn Thr Val Arg Ile Ser Glu Thr 50 55 60 Leu Gln Arg Phe Ala Arg ArgSer Ser His Glu Asn Gly Arg Pro Ser 65 70 75 80 Phe Pro Pro Lys Gln LysArg Lys Met Glu Arg Thr Ile Glu Pro Glu 85 90 95 Val 56 293 DNA Equineinfluenza virus H3N8 56 actttagtgt gattttcgaa aggctggaga cactaatactacttagagcc ttcaccgaag 60 aaggagcagt cgttggcgaa atttcaccat tgccttctcttccaggacat actaatgagg 120 atgtcaaaaa tgcaattggg gtcctcatcg gaggacttaaatggaatgat aatacggtta 180 gaatctctga aactctacag agattcgctc ggagaagcagtcatgagaat gggagacctt 240 cattccctcc aaagcagaaa cgaaaaatgg agagaacaattgagccagaa gtt 293 57 888 DNA Equine influenza virus H3N8 CDS(27)..(716) 57 agcaaaagca gggtgacaaa aacata atg gat tcc aac act gtg tcaagc ttt 53 Met Asp Ser Asn Thr Val Ser Ser Phe 1 5 cag gta gac tgt tttctt tgg cat gtc cgc aaa cga ttt gca gac caa 101 Gln Val Asp Cys Phe LeuTrp His Val Arg Lys Arg Phe Ala Asp Gln 10 15 20 25 gaa ctg ggt gat gcccca ttc ctt gac cgg ctt cgc cga gac cag aag 149 Glu Leu Gly Asp Ala ProPhe Leu Asp Arg Leu Arg Arg Asp Gln Lys 30 35 40 tcc cta aaa gga aga ggtagc act ctt ggt ctg gac atc gaa aca gcc 197 Ser Leu Lys Gly Arg Gly SerThr Leu Gly Leu Asp Ile Glu Thr Ala 45 50 55 act cgt gca gga aag cag atagtg gag cag att ctg gaa gag gaa tca 245 Thr Arg Ala Gly Lys Gln Ile ValGlu Gln Ile Leu Glu Glu Glu Ser 60 65 70 gat gag gca ctt aaa atg acc attgcc tct gtt cct gct tca cgc tac 293 Asp Glu Ala Leu Lys Met Thr Ile AlaSer Val Pro Ala Ser Arg Tyr 75 80 85 tta act gac atg act ctt gat gag atgtca aga gac tgg ttc atg ctc 341 Leu Thr Asp Met Thr Leu Asp Glu Met SerArg Asp Trp Phe Met Leu 90 95 100 105 atg ccc aag cag aaa gta aca ggctcc cta tgt ata aga atg gac cag 389 Met Pro Lys Gln Lys Val Thr Gly SerLeu Cys Ile Arg Met Asp Gln 110 115 120 gca atc atg gat aag aac atc atactt aaa gca aac ttt agt gtg att 437 Ala Ile Met Asp Lys Asn Ile Ile LeuLys Ala Asn Phe Ser Val Ile 125 130 135 ttc gaa agg ctg gag aca cta atacta ctt aga gcc ttc acc gaa gaa 485 Phe Glu Arg Leu Glu Thr Leu Ile LeuLeu Arg Ala Phe Thr Glu Glu 140 145 150 gga gca gtc gtt ggc gaa att tcacca ttg cct tct ctt cca gga cat 533 Gly Ala Val Val Gly Glu Ile Ser ProLeu Pro Ser Leu Pro Gly His 155 160 165 act aat gag gat gtc aaa aat gcaatt ggg gtc ctc atc gga gga ctt 581 Thr Asn Glu Asp Val Lys Asn Ala IleGly Val Leu Ile Gly Gly Leu 170 175 180 185 aaa tgg aat gat aat acg gttaga atc tct gaa act cta cag aga ttc 629 Lys Trp Asn Asp Asn Thr Val ArgIle Ser Glu Thr Leu Gln Arg Phe 190 195 200 gct tgg aga agc agt cat gagaat ggg aga cct tca ttc cct cca aag 677 Ala Trp Arg Ser Ser His Glu AsnGly Arg Pro Ser Phe Pro Pro Lys 205 210 215 cag aaa cga aaa atg gag agaaca att gag cca gaa gtt tgaagaaata 726 Gln Lys Arg Lys Met Glu Arg ThrIle Glu Pro Glu Val 220 225 230 agatggttga ttgaagaagt gcgacatagattgaaaaata cagaaaatag ttttgaacaa 786 ataacattta tgcaagcctt acaactattgcttgaagtag aacaagagat aagaactttc 846 tcgtttcagc ttatttaatg ataaaaaacacccttgtttc ta 888 58 230 PRT Equine influenza virus H3N8 58 Met Asp SerAsn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp 1 5 10 15 His ValArg Lys Arg Phe Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe 20 25 30 Leu AspArg Leu Arg Arg Asp Gln Lys Ser Leu Lys Gly Arg Gly Ser 35 40 45 Thr LeuGly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile 50 55 60 Val GluGln Ile Leu Glu Glu Glu Ser Asp Glu Ala Leu Lys Met Thr 65 70 75 80 IleAla Ser Val Pro Ala Ser Arg Tyr Leu Thr Asp Met Thr Leu Asp 85 90 95 GluMet Ser Arg Asp Trp Phe Met Leu Met Pro Lys Gln Lys Val Thr 100 105 110Gly Ser Leu Cys Ile Arg Met Asp Gln Ala Ile Met Asp Lys Asn Ile 115 120125 Ile Leu Lys Ala Asn Phe Ser Val Ile Phe Glu Arg Leu Glu Thr Leu 130135 140 Ile Leu Leu Arg Ala Phe Thr Glu Glu Gly Ala Val Val Gly Glu Ile145 150 155 160 Ser Pro Leu Pro Ser Leu Pro Gly His Thr Asn Glu Asp ValLys Asn 165 170 175 Ala Ile Gly Val Leu Ile Gly Gly Leu Lys Trp Asn AspAsn Thr Val 180 185 190 Arg Ile Ser Glu Thr Leu Gln Arg Phe Ala Trp ArgSer Ser His Glu 195 200 205 Asn Gly Arg Pro Ser Phe Pro Pro Lys Gln LysArg Lys Met Glu Arg 210 215 220 Thr Ile Glu Pro Glu Val 225 230 59 690DNA Equine influenza virus H3N8 59 atggattcca acactgtgtc aagctttcaggtagactgtt ttctttggca tgtccgcaaa 60 cgatttgcag accaagaact gggtgatgccccattccttg accggcttcg ccgagaccag 120 aagtccctaa aaggaagagg tagcactcttggtctggaca tcgaaacagc cactcgtgca 180 ggaaagcaga tagtggagca gattctggaagaggaatcag atgaggcact taaaatgacc 240 attgcctctg ttcctgcttc acgctacttaactgacatga ctcttgatga gatgtcaaga 300 gactggttca tgctcatgcc caagcagaaagtaacaggct ccctatgtat aagaatggac 360 caggcaatca tggataagaa catcatacttaaagcaaact ttagtgtgat tttcgaaagg 420 ctggagacac taatactact tagagccttcaccgaagaag gagcagtcgt tggcgaaatt 480 tcaccattgc cttctcttcc aggacatactaatgaggatg tcaaaaatgc aattggggtc 540 ctcatcggag gacttaaatg gaatgataatacggttagaa tctctgaaac tctacagaga 600 ttcgcttgga gaagcagtca tgagaatgggagaccttcat tccctccaaa gcagaaacga 660 aaaatggaga gaacaattga gccagaagtt690 60 21 DNA Artificial Sequence Description of Artificial SequenceSynthetic Primer 60 agcaaagcag gtgacaaaaa c 21 61 19 DNA ArtificialSequence Description of Artificial Sequence Synthetic Primer 61agtagaaaca agggtgttt 19 62 1229 DNA Equine influenza virus H3N8 CDS(36)..(1229) 62 gaattcggct tagcaaaagc aggcaaacta tttga atg gat gtc aatccg act 53 Met Asp Val Asn Pro Thr 1 5 cta ctc ttc tta aag gtg cca gcgcaa aat gct ata agc aca aca ttc 101 Leu Leu Phe Leu Lys Val Pro Ala GlnAsn Ala Ile Ser Thr Thr Phe 10 15 20 cct tat act gga gat cct ccc tac agtcat gga aca ggg aca gga tac 149 Pro Tyr Thr Gly Asp Pro Pro Tyr Ser HisGly Thr Gly Thr Gly Tyr 25 30 35 acc atg gat act gtc aac aga aca cat caatac tca gaa aag ggg aaa 197 Thr Met Asp Thr Val Asn Arg Thr His Gln TyrSer Glu Lys Gly Lys 40 45 50 tgg aca aca aac act gag att gga gca cca caactt aat cca atc gat 245 Trp Thr Thr Asn Thr Glu Ile Gly Ala Pro Gln LeuAsn Pro Ile Asp 55 60 65 70 gga ccg ctt cct gaa gac aat gaa cca agt gggtac gcc caa aca gat 293 Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser Gly TyrAla Gln Thr Asp 75 80 85 tgt gta ttg gaa gca atg gct ttc ctt gaa gaa tcccat ccc gga atc 341 Cys Val Leu Glu Ala Met Ala Phe Leu Glu Glu Ser HisPro Gly Ile 90 95 100 ttt gaa aat tcg tgt ctt gaa aca atg gag gtg gttcag cag aca aga 389 Phe Glu Asn Ser Cys Leu Glu Thr Met Glu Val Val GlnGln Thr Arg 105 110 115 gtg gac aaa cta aca caa ggc cga caa act tac gattgg acc ttg aat 437 Val Asp Lys Leu Thr Gln Gly Arg Gln Thr Tyr Asp TrpThr Leu Asn 120 125 130 agg aat caa cct gcc gca aca gca ctt gct aat acaatt gaa gtg ttc 485 Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala Asn Thr IleGlu Val Phe 135 140 145 150 aga tca aat gat ctg act tcc agt gag tca gggaga tta atg gac ttc 533 Arg Ser Asn Asp Leu Thr Ser Ser Glu Ser Gly ArgLeu Met Asp Phe 155 160 165 ctc aaa gat gtc atg gag tcc atg aac aag gaagaa atg gaa ata aca 581 Leu Lys Asp Val Met Glu Ser Met Asn Lys Glu GluMet Glu Ile Thr 170 175 180 aca cac ttc caa cgg aag aga aga gta aga gacaac atg aca aag aga 629 Thr His Phe Gln Arg Lys Arg Arg Val Arg Asp AsnMet Thr Lys Arg 185 190 195 atg gtg aca cag aga acc ata ggg aag aaa aaacaa cga tta aac aga 677 Met Val Thr Gln Arg Thr Ile Gly Lys Lys Lys GlnArg Leu Asn Arg 200 205 210 aag agc tat ctg atc agg gca tta acc tta aacaca atg acc aag gac 725 Lys Ser Tyr Leu Ile Arg Ala Leu Thr Leu Asn ThrMet Thr Lys Asp 215 220 225 230 gct gag aga ggg aaa ttg aaa cga cga gcaatt gca acc cca gga atg 773 Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala IleAla Thr Pro Gly Met 235 240 245 cag ata aga ggg ttt gta tat ttt gtt gaaaca tta gcc cga aga ata 821 Gln Ile Arg Gly Phe Val Tyr Phe Val Glu ThrLeu Ala Arg Arg Ile 250 255 260 tgt gaa aag ctt gaa caa tca gga ttg ccagtt ggc ggt aat gag aaa 869 Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro ValGly Gly Asn Glu Lys 265 270 275 aag gcc aaa ctg gct aat gtc gtc aga aaaatg atg act aat tcc caa 917 Lys Ala Lys Leu Ala Asn Val Val Arg Lys MetMet Thr Asn Ser Gln 280 285 290 gac act gaa ctc tcc ttc acc atc act ggggac aat acc aaa tgg aat 965 Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly AspAsn Thr Lys Trp Asn 295 300 305 310 gaa aat cag aac cca cgc atg ttc ctggca atg atc aca tac ata act 1013 Glu Asn Gln Asn Pro Arg Met Phe Leu AlaMet Ile Thr Tyr Ile Thr 315 320 325 aga aac cag cca gaa tgg ttc aga aatgtt cta agc att gca ccg att 1061 Arg Asn Gln Pro Glu Trp Phe Arg Asn ValLeu Ser Ile Ala Pro Ile 330 335 340 atg ttc tca aat aaa atg gca aga ctgggg aaa gga tat atg ttt gaa 1109 Met Phe Ser Asn Lys Met Ala Arg Leu GlyLys Gly Tyr Met Phe Glu 345 350 355 agc aaa agt atg aaa ttg aga act caaata cca gca gaa atg ctc gca 1157 Ser Lys Ser Met Lys Leu Arg Thr Gln IlePro Ala Glu Met Leu Ala 360 365 370 agc att gat ctg aaa tat ttc aat gattca aca aaa aag aaa att gag 1205 Ser Ile Asp Leu Lys Tyr Phe Asn Asp SerThr Lys Lys Lys Ile Glu 375 380 385 390 aag ata cga cca caa gcc gaa ttc1229 Lys Ile Arg Pro Gln Ala Glu Phe 395 63 398 PRT Equine influenzavirus H3N8 63 Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro AlaGln Asn 1 5 10 15 Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro ProTyr Ser His 20 25 30 Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn ArgThr His Gln 35 40 45 Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu IleGly Ala Pro 50 55 60 Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp AsnGlu Pro Ser 65 70 75 80 Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu Ala MetAla Phe Leu Glu 85 90 95 Glu Ser His Pro Gly Ile Phe Glu Asn Ser Cys LeuGlu Thr Met Glu 100 105 110 Val Val Gln Gln Thr Arg Val Asp Lys Leu ThrGln Gly Arg Gln Thr 115 120 125 Tyr Asp Trp Thr Leu Asn Arg Asn Gln ProAla Ala Thr Ala Leu Ala 130 135 140 Asn Thr Ile Glu Val Phe Arg Ser AsnAsp Leu Thr Ser Ser Glu Ser 145 150 155 160 Gly Arg Leu Met Asp Phe LeuLys Asp Val Met Glu Ser Met Asn Lys 165 170 175 Glu Glu Met Glu Ile ThrThr His Phe Gln Arg Lys Arg Arg Val Arg 180 185 190 Asp Asn Met Thr LysArg Met Val Thr Gln Arg Thr Ile Gly Lys Lys 195 200 205 Lys Gln Arg LeuAsn Arg Lys Ser Tyr Leu Ile Arg Ala Leu Thr Leu 210 215 220 Asn Thr MetThr Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala 225 230 235 240 IleAla Thr Pro Gly Met Gln Ile Arg Gly Phe Val Tyr Phe Val Glu 245 250 255Thr Leu Ala Arg Arg Ile Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro 260 265270 Val Gly Gly Asn Glu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys 275280 285 Met Met Thr Asn Ser Gln Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly290 295 300 Asp Asn Thr Lys Trp Asn Glu Asn Gln Asn Pro Arg Met Phe LeuAla 305 310 315 320 Met Ile Thr Tyr Ile Thr Arg Asn Gln Pro Glu Trp PheArg Asn Val 325 330 335 Leu Ser Ile Ala Pro Ile Met Phe Ser Asn Lys MetAla Arg Leu Gly 340 345 350 Lys Gly Tyr Met Phe Glu Ser Lys Ser Met LysLeu Arg Thr Gln Ile 355 360 365 Pro Ala Glu Met Leu Ala Ser Ile Asp LeuLys Tyr Phe Asn Asp Ser 370 375 380 Thr Lys Lys Lys Ile Glu Lys Ile ArgPro Gln Ala Glu Phe 385 390 395 64 1194 DNA Equine influenza virus H3N864 atggatgtca atccgactct actcttctta aaggtgccag cgcaaaatgc tataagcaca 60acattccctt atactggaga tcctccctac agtcatggaa cagggacagg atacaccatg 120gatactgtca acagaacaca tcaatactca gaaaagggga aatggacaac aaacactgag 180attggagcac cacaacttaa tccaatcgat ggaccgcttc ctgaagacaa tgaaccaagt 240gggtacgccc aaacagattg tgtattggaa gcaatggctt tccttgaaga atcccatccc 300ggaatctttg aaaattcgtg tcttgaaaca atggaggtgg ttcagcagac aagagtggac 360aaactaacac aaggccgaca aacttacgat tggaccttga ataggaatca acctgccgca 420acagcacttg ctaatacaat tgaagtgttc agatcaaatg atctgacttc cagtgagtca 480gggagattaa tggacttcct caaagatgtc atggagtcca tgaacaagga agaaatggaa 540ataacaacac acttccaacg gaagagaaga gtaagagaca acatgacaaa gagaatggtg 600acacagagaa ccatagggaa gaaaaaacaa cgattaaaca gaaagagcta tctgatcagg 660gcattaacct taaacacaat gaccaaggac gctgagagag ggaaattgaa acgacgagca 720attgcaaccc caggaatgca gataagaggg tttgtatatt ttgttgaaac attagcccga 780agaatatgtg aaaagcttga acaatcagga ttgccagttg gcggtaatga gaaaaaggcc 840aaactggcta atgtcgtcag aaaaatgatg actaattccc aagacactga actctccttc 900accatcactg gggacaatac caaatggaat gaaaatcaga acccacgcat gttcctggca 960atgatcacat acataactag aaaccagcca gaatggttca gaaatgttct aagcattgca 1020ccgattatgt tctcaaataa aatggcaaga ctggggaaag gatatatgtt tgaaagcaaa 1080agtatgaaat tgagaactca aataccagca gaaatgctcg caagcattga tctgaaatat 1140ttcaatgatt caacaaaaaa gaaaattgag aagatacgac cacaagccga attc 1194 65 673DNA Equine influenza virus H3N8 CDS (36)..(671) 65 gaattcggct tagcaaaagcaggcaaacta tttga atg gat gtc aat ccg act 53 Met Asp Val Asn Pro Thr 1 5cta ctc ttc tta aag gtg cca gcg caa aat gct ata agc aca aca ttc 101 LeuLeu Phe Leu Lys Val Pro Ala Gln Asn Ala Ile Ser Thr Thr Phe 10 15 20 ccttat act gga gat cct ccc tac agt cat gga aca ggg aca gga tac 149 Pro TyrThr Gly Asp Pro Pro Tyr Ser His Gly Thr Gly Thr Gly Tyr 25 30 35 acc atggat act gtc aac aga aca cat caa tac tca gaa aag ggg aaa 197 Thr Met AspThr Val Asn Arg Thr His Gln Tyr Ser Glu Lys Gly Lys 40 45 50 tgg aca acaaac act gag att gga gca cca caa ctt aat cca atc gat 245 Trp Thr Thr AsnThr Glu Ile Gly Ala Pro Gln Leu Asn Pro Ile Asp 55 60 65 70 gga ccg cttcct gaa gac aat gaa cca agt ggg tac gcc caa aca gat 293 Gly Pro Leu ProGlu Asp Asn Glu Pro Ser Gly Tyr Ala Gln Thr Asp 75 80 85 tgt gta ttg gaagca atg gct ttc ctt gaa gaa tcc cat ccc gga atc 341 Cys Val Leu Glu AlaMet Ala Phe Leu Glu Glu Ser His Pro Gly Ile 90 95 100 ttt gaa aat tcgtgt ctt gaa aca atg gag gtg gtt cag cag aca aga 389 Phe Glu Asn Ser CysLeu Glu Thr Met Glu Val Val Gln Gln Thr Arg 105 110 115 gtg gac aaa ctaaca caa ggc cga caa act tac gat tgg acc ttg aat 437 Val Asp Lys Leu ThrGln Gly Arg Gln Thr Tyr Asp Trp Thr Leu Asn 120 125 130 agg aat caa cctgcc gca aca gca ctt gct aat aca att gaa gtg ttc 485 Arg Asn Gln Pro AlaAla Thr Ala Leu Ala Asn Thr Ile Glu Val Phe 135 140 145 150 aga tca aatgat ctg act tcc agt gag tca ggg aga tta atg gac ttc 533 Arg Ser Asn AspLeu Thr Ser Ser Glu Ser Gly Arg Leu Met Asp Phe 155 160 165 ctc aaa gatgtc atg gag tcc atg aac aag gaa gaa atg gaa ata aca 581 Leu Lys Asp ValMet Glu Ser Met Asn Lys Glu Glu Met Glu Ile Thr 170 175 180 aca cac ttccaa cgg aag aga aga gta aga gac aac atg aca aag aga 629 Thr His Phe GlnArg Lys Arg Arg Val Arg Asp Asn Met Thr Lys Arg 185 190 195 atg gtg acacag aga acc ata ggg aag aaa aaa caa cga tta aa 673 Met Val Thr Gln ArgThr Ile Gly Lys Lys Lys Gln Arg Leu 200 205 210 66 212 PRT Equineinfluenza virus H3N8 66 Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys ValPro Ala Gln Asn 1 5 10 15 Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly AspPro Pro Tyr Ser His 20 25 30 Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr ValAsn Arg Thr His Gln 35 40 45 Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn ThrGlu Ile Gly Ala Pro 50 55 60 Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro GluAsp Asn Glu Pro Ser 65 70 75 80 Gly Tyr Ala Gln Thr Asp Cys Val Leu GluAla Met Ala Phe Leu Glu 85 90 95 Glu Ser His Pro Gly Ile Phe Glu Asn SerCys Leu Glu Thr Met Glu 100 105 110 Val Val Gln Gln Thr Arg Val Asp LysLeu Thr Gln Gly Arg Gln Thr 115 120 125 Tyr Asp Trp Thr Leu Asn Arg AsnGln Pro Ala Ala Thr Ala Leu Ala 130 135 140 Asn Thr Ile Glu Val Phe ArgSer Asn Asp Leu Thr Ser Ser Glu Ser 145 150 155 160 Gly Arg Leu Met AspPhe Leu Lys Asp Val Met Glu Ser Met Asn Lys 165 170 175 Glu Glu Met GluIle Thr Thr His Phe Gln Arg Lys Arg Arg Val Arg 180 185 190 Asp Asn MetThr Lys Arg Met Val Thr Gln Arg Thr Ile Gly Lys Lys 195 200 205 Lys GlnArg Leu 210 67 636 DNA Equine influenza virus H3N8 67 atggatgtcaatccgactct actcttctta aaggtgccag cgcaaaatgc tataagcaca 60 acattcccttatactggaga tcctccctac agtcatggaa cagggacagg atacaccatg 120 gatactgtcaacagaacaca tcaatactca gaaaagggga aatggacaac aaacactgag 180 attggagcaccacaacttaa tccaatcgat ggaccgcttc ctgaagacaa tgaaccaagt 240 gggtacgcccaaacagattg tgtattggaa gcaatggctt tccttgaaga atcccatccc 300 ggaatctttgaaaattcgtg tcttgaaaca atggaggtgg ttcagcagac aagagtggac 360 aaactaacacaaggccgaca aacttacgat tggaccttga ataggaatca acctgccgca 420 acagcacttgctaatacaat tgaagtgttc agatcaaatg atctgacttc cagtgagtca 480 gggagattaatggacttcct caaagatgtc atggagtcca tgaacaagga agaaatggaa 540 ataacaacacacttccaacg gaagagaaga gtaagagaca acatgacaaa gagaatggtg 600 acacagagaaccatagggaa gaaaaaacaa cgatta 636 68 1225 DNA Equine influenza virus H3N8CDS (34)..(1218) 68 gaattcagga gcaaaagcag gcaaactatt tga atg gat gtc aatccg act cta 54 Met Asp Val Asn Pro Thr Leu 1 5 ctc ttc tta aag gtg ccagcg caa aat gct ata agc aca aca ttc cct 102 Leu Phe Leu Lys Val Pro AlaGln Asn Ala Ile Ser Thr Thr Phe Pro 10 15 20 tat act gga gat cct ccc tacagt cat gga aca ggg aca gga tac acc 150 Tyr Thr Gly Asp Pro Pro Tyr SerHis Gly Thr Gly Thr Gly Tyr Thr 25 30 35 atg gat act gtc aac aga aca catcaa tac tca gaa aag ggg aaa tgg 198 Met Asp Thr Val Asn Arg Thr His GlnTyr Ser Glu Lys Gly Lys Trp 40 45 50 55 aca aca aac act gag att gga gcacca caa ctt aat cca atc gat gga 246 Thr Thr Asn Thr Glu Ile Gly Ala ProGln Leu Asn Pro Ile Asp Gly 60 65 70 ccg ctt cct gaa gac aat gaa cca agtggg tac gcc caa aca gat tgt 294 Pro Leu Pro Glu Asp Asn Glu Pro Ser GlyTyr Ala Gln Thr Asp Cys 75 80 85 gta ttg gaa gca atg gct ttc ctt gaa gaatcc cat ccc gga atc ttt 342 Val Leu Glu Ala Met Ala Phe Leu Glu Glu SerHis Pro Gly Ile Phe 90 95 100 gaa aat tcg tgt ctt gaa aca atg gag gtggtt cag cag aca aga gtg 390 Glu Asn Ser Cys Leu Glu Thr Met Glu Val ValGln Gln Thr Arg Val 105 110 115 gac aaa cta aca caa ggc cga caa act tacgat tgg acc ttg aat agg 438 Asp Lys Leu Thr Gln Gly Arg Gln Thr Tyr AspTrp Thr Leu Asn Arg 120 125 130 135 aat caa cct gcc gca aca gca ctt gctaat aca att gaa gtg ttc aga 486 Asn Gln Pro Ala Ala Thr Ala Leu Ala AsnThr Ile Glu Val Phe Arg 140 145 150 tca aat gat ctg act tcc agt gag tcaggg aga tta atg gac ttc ctc 534 Ser Asn Asp Leu Thr Ser Ser Glu Ser GlyArg Leu Met Asp Phe Leu 155 160 165 aaa gat gtc atg gag tcc atg aac aaggaa gaa atg gaa ata aca aca 582 Lys Asp Val Met Glu Ser Met Asn Lys GluGlu Met Glu Ile Thr Thr 170 175 180 cac ttc caa cgg aag aga aga gta agagac aac atg aca aag aga atg 630 His Phe Gln Arg Lys Arg Arg Val Arg AspAsn Met Thr Lys Arg Met 185 190 195 gtg aca cag aga acc ata ggg aag aaaaaa caa cga tta aac aga aag 678 Val Thr Gln Arg Thr Ile Gly Lys Lys LysGln Arg Leu Asn Arg Lys 200 205 210 215 agc tat ctg atc agg gca tta acctta aac aca atg acc aag gac gct 726 Ser Tyr Leu Ile Arg Ala Leu Thr LeuAsn Thr Met Thr Lys Asp Ala 220 225 230 gag aga ggg aaa ttg aaa cga cgagca att gca acc cca gga atg cag 774 Glu Arg Gly Lys Leu Lys Arg Arg AlaIle Ala Thr Pro Gly Met Gln 235 240 245 ata aga ggg ttt gta tat ttt gttgaa aca tta gcc cga aga ata tgt 822 Ile Arg Gly Phe Val Tyr Phe Val GluThr Leu Ala Arg Arg Ile Cys 250 255 260 gaa aag ctt gaa caa tca gga ttgcca gtt ggc ggt aat gag aaa aag 870 Glu Lys Leu Glu Gln Ser Gly Leu ProVal Gly Gly Asn Glu Lys Lys 265 270 275 gcc aaa ctg gct aat gtc gtc agaaaa atg atg act aat tcc caa gac 918 Ala Lys Leu Ala Asn Val Val Arg LysMet Met Thr Asn Ser Gln Asp 280 285 290 295 act gaa ctc tcc ttc acc atcact ggg gac aat acc aaa tgg aat gaa 966 Thr Glu Leu Ser Phe Thr Ile ThrGly Asp Asn Thr Lys Trp Asn Glu 300 305 310 aat cag aac cca cgc atg ttcctg gca atg atc aca tac ata act aga 1014 Asn Gln Asn Pro Arg Met Phe LeuAla Met Ile Thr Tyr Ile Thr Arg 315 320 325 aac cag cca gaa tgg ttc agaaat gtt cta agc att gca ccg att atg 1062 Asn Gln Pro Glu Trp Phe Arg AsnVal Leu Ser Ile Ala Pro Ile Met 330 335 340 ttc tca aat aaa atg gca agactg ggg aaa gga tat atg ttt gaa agc 1110 Phe Ser Asn Lys Met Ala Arg LeuGly Lys Gly Tyr Met Phe Glu Ser 345 350 355 aaa agt atg aaa ttg aga actcaa ata cca gca gaa atg ctc gca agc 1158 Lys Ser Met Lys Leu Arg Thr GlnIle Pro Ala Glu Met Leu Ala Ser 360 365 370 375 att gat ctg aaa tat ttcaat gat tca aca aaa aag aaa att gag aag 1206 Ile Asp Leu Lys Tyr Phe AsnAsp Ser Thr Lys Lys Lys Ile Glu Lys 380 385 390 ata cga cca ccc tgaattc1225 Ile Arg Pro Pro 395 69 395 PRT Equine influenza virus H3N8 69 MetAsp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala Gln Asn 1 5 10 15Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His 20 25 30Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His Gln 35 40 45Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu Ile Gly Ala Pro 50 55 60Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser 65 70 7580 Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu 85 9095 Glu Ser His Pro Gly Ile Phe Glu Asn Ser Cys Leu Glu Thr Met Glu 100105 110 Val Val Gln Gln Thr Arg Val Asp Lys Leu Thr Gln Gly Arg Gln Thr115 120 125 Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala LeuAla 130 135 140 Asn Thr Ile Glu Val Phe Arg Ser Asn Asp Leu Thr Ser SerGlu Ser 145 150 155 160 Gly Arg Leu Met Asp Phe Leu Lys Asp Val Met GluSer Met Asn Lys 165 170 175 Glu Glu Met Glu Ile Thr Thr His Phe Gln ArgLys Arg Arg Val Arg 180 185 190 Asp Asn Met Thr Lys Arg Met Val Thr GlnArg Thr Ile Gly Lys Lys 195 200 205 Lys Gln Arg Leu Asn Arg Lys Ser TyrLeu Ile Arg Ala Leu Thr Leu 210 215 220 Asn Thr Met Thr Lys Asp Ala GluArg Gly Lys Leu Lys Arg Arg Ala 225 230 235 240 Ile Ala Thr Pro Gly MetGln Ile Arg Gly Phe Val Tyr Phe Val Glu 245 250 255 Thr Leu Ala Arg ArgIle Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro 260 265 270 Val Gly Gly AsnGlu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys 275 280 285 Met Met ThrAsn Ser Gln Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly 290 295 300 Asp AsnThr Lys Trp Asn Glu Asn Gln Asn Pro Arg Met Phe Leu Ala 305 310 315 320Met Ile Thr Tyr Ile Thr Arg Asn Gln Pro Glu Trp Phe Arg Asn Val 325 330335 Leu Ser Ile Ala Pro Ile Met Phe Ser Asn Lys Met Ala Arg Leu Gly 340345 350 Lys Gly Tyr Met Phe Glu Ser Lys Ser Met Lys Leu Arg Thr Gln Ile355 360 365 Pro Ala Glu Met Leu Ala Ser Ile Asp Leu Lys Tyr Phe Asn AspSer 370 375 380 Thr Lys Lys Lys Ile Glu Lys Ile Arg Pro Pro 385 390 39570 1185 DNA Equine influenza virus H3N8 70 atggatgtca atccgactctactcttctta aaggtgccag cgcaaaatgc tataagcaca 60 acattccctt atactggagatcctccctac agtcatggaa cagggacagg atacaccatg 120 gatactgtca acagaacacatcaatactca gaaaagggga aatggacaac aaacactgag 180 attggagcac cacaacttaatccaatcgat ggaccgcttc ctgaagacaa tgaaccaagt 240 gggtacgccc aaacagattgtgtattggaa gcaatggctt tccttgaaga atcccatccc 300 ggaatctttg aaaattcgtgtcttgaaaca atggaggtgg ttcagcagac aagagtggac 360 aaactaacac aaggccgacaaacttacgat tggaccttga ataggaatca acctgccgca 420 acagcacttg ctaatacaattgaagtgttc agatcaaatg atctgacttc cagtgagtca 480 gggagattaa tggacttcctcaaagatgtc atggagtcca tgaacaagga agaaatggaa 540 ataacaacac acttccaacggaagagaaga gtaagagaca acatgacaaa gagaatggtg 600 acacagagaa ccatagggaagaaaaaacaa cgattaaaca gaaagagcta tctgatcagg 660 gcattaacct taaacacaatgaccaaggac gctgagagag ggaaattgaa acgacgagca 720 attgcaaccc caggaatgcagataagaggg tttgtatatt ttgttgaaac attagcccga 780 agaatatgtg aaaagcttgaacaatcagga ttgccagttg gcggtaatga gaaaaaggcc 840 aaactggcta atgtcgtcagaaaaatgatg actaattccc aagacactga actctccttc 900 accatcactg gggacaataccaaatggaat gaaaatcaga acccacgcat gttcctggca 960 atgatcacat acataactagaaaccagcca gaatggttca gaaatgttct aagcattgca 1020 ccgattatgt tctcaaataaaatggcaaga ctggggaaag gatatatgtt tgaaagcaaa 1080 agtatgaaat tgagaactcaaataccagca gaaatgctcg caagcattga tctgaaatat 1140 ttcaatgatt caacaaaaaagaaaattgag aagatacgac caccc 1185 71 1221 DNA Equine influenza virus H3N871 gaattcagga aagcaggcaa actatttgaa tggatgtcaa tccgactcta ctcttcttaa 60aggtgccagc gcaaaatgct ataagcacaa cattccctta tactggagat cctccctaca 120gtcatggaac agggacagga tacaccatgg atactgtcaa cagaacacat caatactcag 180aaaaggggaa atggacaaca aacactgaga ttggagcacc acaacttaat ccaatcgatg 240gaccgcttcc tgaagacaat gaaccaagtg ggtacgccca aacagattgt gtattggaag 300caatggcttt ccttgaagaa tcccatcccg gaatctttga aaattcgtgt cttgaaacaa 360tggaggtggt tcagcagaca agagtggaca aactaacaca aggccgacaa acttacgatt 420ggaccttgaa taggaatcaa cctgccgcaa cagcacttgc taatacaatt gaagtgttca 480gatcaaatga tctgacttcc agtgagtcag ggagattaat ggacttcctc aaagatgtca 540tggagtccat gaacaaggaa gaaatggaaa taacaacaca cttccaacgg aagagaagag 600taagagacaa catgacaaag agaatggtga cacagagaac catagggaag aaaaaacaac 660gattaaacag aaagagctat ctgatcaggg cattaacctt aaacacaatg accaaggacg 720ctgagagagg gaaattgaaa cgacgagcaa ttgcaacccc aggaatgcag ataagagggt 780ttgtatattt tgttgaaaca ttagcccgaa gaatatgtga aaagcttgaa caatcaggat 840tgccagttgg cggtaatgag aaaaaggcca aactggctaa tgtcgtcaga aaaatgatga 900ctaattccca agacactgaa ctctccttca ccatcactgg ggacaatacc aaatggaatg 960aaaatcagaa cccacgcatg ttcctggcaa tgatcacata cataactaga aaccagccag 1020aatggttcag aaatgttcta agcattgcac cgattatgtt ctcaaataaa atggcaagac 1080tggggaaagg atatatgttt gaaagcaaaa gtatgaaatt gagaactcaa ataccagcag 1140aaatgctcgc aagcattgat ctgaaatatt tcaatgattc aacaaaaaag aaaattgaga 1200agatacgacc accctgaatt c 1221 72 18 DNA Artificial Sequence Descriptionof Artificial Sequence Synthetic Primer 72 gcaaatgcag gaccaaag 18 73 18DNA Artificial Sequence Description of Artificial Sequence SyntheticPrimer 73 gactgaggac tcagcttc 18 74 19 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Primer 74 caatatcctccccaatttc 19 75 19 DNA Artificial Sequence Description of ArtificialSequence Synthetic Primer 75 ggaaggtttg caggacctt 19 76 1228 DNA Equineinfluenza virus H3N8 CDS (3)..(1166) 76 gg ggc ggg tac cca aac tat ctccaa gct tgg aag caa gta tta gca 47 Gly Gly Tyr Pro Asn Tyr Leu Gln AlaTrp Lys Gln Val Leu Ala 1 5 10 15 gaa cta caa gac ctt gag aac gaa gaaaag acc cct aag acc aag aat 95 Glu Leu Gln Asp Leu Glu Asn Glu Glu LysThr Pro Lys Thr Lys Asn 20 25 30 atg aaa aaa aca agc caa ttg aaa tgg gcactc ggt gaa aat atg gca 143 Met Lys Lys Thr Ser Gln Leu Lys Trp Ala LeuGly Glu Asn Met Ala 35 40 45 cca gag aaa gtg gat ttt gag gat tgt aaa gacatc aat gat ttg aaa 191 Pro Glu Lys Val Asp Phe Glu Asp Cys Lys Asp IleAsn Asp Leu Lys 50 55 60 cag tat gac agt gat gag cca gaa aca agg tct cttgca agt tgg att 239 Gln Tyr Asp Ser Asp Glu Pro Glu Thr Arg Ser Leu AlaSer Trp Ile 65 70 75 caa agt gag ttc aac aaa gct tgt gag ctg aca gat tcaagc tgg ata 287 Gln Ser Glu Phe Asn Lys Ala Cys Glu Leu Thr Asp Ser SerTrp Ile 80 85 90 95 gag ctc gat gaa att ggg gag gat att gcc cca ata gaatac att gcg 335 Glu Leu Asp Glu Ile Gly Glu Asp Ile Ala Pro Ile Glu TyrIle Ala 100 105 110 agc atg agg aga aat tat ttt act gct gag gtt tcc cattgt aga gca 383 Ser Met Arg Arg Asn Tyr Phe Thr Ala Glu Val Ser His CysArg Ala 115 120 125 aca gaa tat ata atg aag gga gtg tac atc aac act gctcta ctc aat 431 Thr Glu Tyr Ile Met Lys Gly Val Tyr Ile Asn Thr Ala LeuLeu Asn 130 135 140 gca tcc tgt gct gcg atg gat gaa ttc caa tta att ccgatg ata agc 479 Ala Ser Cys Ala Ala Met Asp Glu Phe Gln Leu Ile Pro MetIle Ser 145 150 155 aaa tgc agg acc aaa gaa ggg aga agg aag aca aat ttatat gga ttc 527 Lys Cys Arg Thr Lys Glu Gly Arg Arg Lys Thr Asn Leu TyrGly Phe 160 165 170 175 ata ata aag gga agg tcc cat tta agg aat gat accgac gtg gta aac 575 Ile Ile Lys Gly Arg Ser His Leu Arg Asn Asp Thr AspVal Val Asn 180 185 190 ttt gta agt atg gaa ttt tct ctc act gat cca agattt gag cca cat 623 Phe Val Ser Met Glu Phe Ser Leu Thr Asp Pro Arg PheGlu Pro His 195 200 205 aaa tgg gaa aaa tac tgc gtt cta gaa att gga gacatg ctc cta agg 671 Lys Trp Glu Lys Tyr Cys Val Leu Glu Ile Gly Asp MetLeu Leu Arg 210 215 220 act gct gta ggt caa gtg tca aga ccc atg ttt ttgtat gta agg aca 719 Thr Ala Val Gly Gln Val Ser Arg Pro Met Phe Leu TyrVal Arg Thr 225 230 235 aat gga acc tct aaa att aaa atg aaa cgg gga atggaa atg aga cgc 767 Asn Gly Thr Ser Lys Ile Lys Met Lys Arg Gly Met GluMet Arg Arg 240 245 250 255 tgc ctc ctt cag tct ctg caa cag att gaa agcatg atc gaa gct gag 815 Cys Leu Leu Gln Ser Leu Gln Gln Ile Glu Ser MetIle Glu Ala Glu 260 265 270 tcc tca gtc aaa gaa aag gac atg acc aaa gaattc ttt gag aac aaa 863 Ser Ser Val Lys Glu Lys Asp Met Thr Lys Glu PhePhe Glu Asn Lys 275 280 285 tca gag aca tgg cct ata gga gag tcc ccc aaagga gtg gaa gag ggc 911 Ser Glu Thr Trp Pro Ile Gly Glu Ser Pro Lys GlyVal Glu Glu Gly 290 295 300 tca atc ggg aag gtt tgc agg acc tta tta gcaaaa tct gtg ttt aac 959 Ser Ile Gly Lys Val Cys Arg Thr Leu Leu Ala LysSer Val Phe Asn 305 310 315 agt ttg tat gca tct cca caa ctg gaa ggg ttttca gct gaa tct agg 1007 Ser Leu Tyr Ala Ser Pro Gln Leu Glu Gly Phe SerAla Glu Ser Arg 320 325 330 335 aaa tta ctt ctc att gtt cag gcc ctt agggat aac ctg gaa cct gga 1055 Lys Leu Leu Leu Ile Val Gln Ala Leu Arg AspAsn Leu Glu Pro Gly 340 345 350 acc ttt gat att ggg ggg tta tat gaa tcaatt gag gag tgc ctg att 1103 Thr Phe Asp Ile Gly Gly Leu Tyr Glu Ser IleGlu Glu Cys Leu Ile 355 360 365 aat gat ccc tgg gtt ttg ctc aat gca tcttgg ttc aac tcc ttc ctt 1151 Asn Asp Pro Trp Val Leu Leu Asn Ala Ser TrpPhe Asn Ser Phe Leu 370 375 380 aca cat gca ctg aag tagttgtagcaatgctacta tttgctatcc atactgtcca 1206 Thr His Ala Leu Lys 385 aaaaagtactcgagccccca ag 1228 77 388 PRT Equine influenza virus H3N8 77 Gly Gly TyrPro Asn Tyr Leu Gln Ala Trp Lys Gln Val Leu Ala Glu 1 5 10 15 Leu GlnAsp Leu Glu Asn Glu Glu Lys Thr Pro Lys Thr Lys Asn Met 20 25 30 Lys LysThr Ser Gln Leu Lys Trp Ala Leu Gly Glu Asn Met Ala Pro 35 40 45 Glu LysVal Asp Phe Glu Asp Cys Lys Asp Ile Asn Asp Leu Lys Gln 50 55 60 Tyr AspSer Asp Glu Pro Glu Thr Arg Ser Leu Ala Ser Trp Ile Gln 65 70 75 80 SerGlu Phe Asn Lys Ala Cys Glu Leu Thr Asp Ser Ser Trp Ile Glu 85 90 95 LeuAsp Glu Ile Gly Glu Asp Ile Ala Pro Ile Glu Tyr Ile Ala Ser 100 105 110Met Arg Arg Asn Tyr Phe Thr Ala Glu Val Ser His Cys Arg Ala Thr 115 120125 Glu Tyr Ile Met Lys Gly Val Tyr Ile Asn Thr Ala Leu Leu Asn Ala 130135 140 Ser Cys Ala Ala Met Asp Glu Phe Gln Leu Ile Pro Met Ile Ser Lys145 150 155 160 Cys Arg Thr Lys Glu Gly Arg Arg Lys Thr Asn Leu Tyr GlyPhe Ile 165 170 175 Ile Lys Gly Arg Ser His Leu Arg Asn Asp Thr Asp ValVal Asn Phe 180 185 190 Val Ser Met Glu Phe Ser Leu Thr Asp Pro Arg PheGlu Pro His Lys 195 200 205 Trp Glu Lys Tyr Cys Val Leu Glu Ile Gly AspMet Leu Leu Arg Thr 210 215 220 Ala Val Gly Gln Val Ser Arg Pro Met PheLeu Tyr Val Arg Thr Asn 225 230 235 240 Gly Thr Ser Lys Ile Lys Met LysArg Gly Met Glu Met Arg Arg Cys 245 250 255 Leu Leu Gln Ser Leu Gln GlnIle Glu Ser Met Ile Glu Ala Glu Ser 260 265 270 Ser Val Lys Glu Lys AspMet Thr Lys Glu Phe Phe Glu Asn Lys Ser 275 280 285 Glu Thr Trp Pro IleGly Glu Ser Pro Lys Gly Val Glu Glu Gly Ser 290 295 300 Ile Gly Lys ValCys Arg Thr Leu Leu Ala Lys Ser Val Phe Asn Ser 305 310 315 320 Leu TyrAla Ser Pro Gln Leu Glu Gly Phe Ser Ala Glu Ser Arg Lys 325 330 335 LeuLeu Leu Ile Val Gln Ala Leu Arg Asp Asn Leu Glu Pro Gly Thr 340 345 350Phe Asp Ile Gly Gly Leu Tyr Glu Ser Ile Glu Glu Cys Leu Ile Asn 355 360365 Asp Pro Trp Val Leu Leu Asn Ala Ser Trp Phe Asn Ser Phe Leu Thr 370375 380 His Ala Leu Lys 385 78 1164 DNA Equine influenza virus H3N8 78ggcgggtacc caaactatct ccaagcttgg aagcaagtat tagcagaact acaagacctt 60gagaacgaag aaaagacccc taagaccaag aatatgaaaa aaacaagcca attgaaatgg 120gcactcggtg aaaatatggc accagagaaa gtggattttg aggattgtaa agacatcaat 180gatttgaaac agtatgacag tgatgagcca gaaacaaggt ctcttgcaag ttggattcaa 240agtgagttca acaaagcttg tgagctgaca gattcaagct ggatagagct cgatgaaatt 300ggggaggata ttgccccaat agaatacatt gcgagcatga ggagaaatta ttttactgct 360gaggtttccc attgtagagc aacagaatat ataatgaagg gagtgtacat caacactgct 420ctactcaatg catcctgtgc tgcgatggat gaattccaat taattccgat gataagcaaa 480tgcaggacca aagaagggag aaggaagaca aatttatatg gattcataat aaagggaagg 540tcccatttaa ggaatgatac cgacgtggta aactttgtaa gtatggaatt ttctctcact 600gatccaagat ttgagccaca taaatgggaa aaatactgcg ttctagaaat tggagacatg 660ctcctaagga ctgctgtagg tcaagtgtca agacccatgt ttttgtatgt aaggacaaat 720ggaacctcta aaattaaaat gaaacgggga atggaaatga gacgctgcct ccttcagtct 780ctgcaacaga ttgaaagcat gatcgaagct gagtcctcag tcaaagaaaa ggacatgacc 840aaagaattct ttgagaacaa atcagagaca tggcctatag gagagtcccc caaaggagtg 900gaagagggct caatcgggaa ggtttgcagg accttattag caaaatctgt gtttaacagt 960ttgtatgcat ctccacaact ggaagggttt tcagctgaat ctaggaaatt acttctcatt 1020gttcaggccc ttagggataa cctggaacct ggaacctttg atattggggg gttatatgaa 1080tcaattgagg agtgcctgat taatgatccc tgggttttgc tcaatgcatc ttggttcaac 1140tccttcctta cacatgcact gaag 1164 79 1223 DNA Equine influenza virus H3N879 ggggcgggta cccaaactat ctccaagctt ggaagcaagt attagcagaa ctacaagacc 60ttgagaacga agaaaagacc cctaagacca agaatatgaa aaaaacaagc caattgaaat 120gggcactcgg tgaaaatatg gcaccagaga aagtggattt tgaggattgt aaagacatca 180atgatttgaa acagtatgac agtgatgagc cagaaacaag gtctcttgca agttggattc 240aaagtgagtt caacaaagct tgtgagctga cagattcaag ctggatagag ctcgatgaaa 300ttggggagga tattgcccca atagaataca ttgcgagcat gaggagaaat tattttactg 360ctgaggtttc ccattgtaga gcaacagaat atataatgaa gggagtgtac atcaacactg 420ctctactcaa tgcatcctgt gctgcgatgg atgaattcca attaattccg atgataagca 480aatgcaggac caaagaaggg agaaggaaga caaatttata tggattcata ataaagggaa 540ggtcccattt aaggaatgat accgacgtgg taaactttgt aagtatggaa ttttctctca 600ctgatccaag atttgagcca cataaatggg aaaaatactg cgttctagaa attggagaca 660tgctcctaag gactgctgta ggtcaagtgt caagacccat gtttttgtat gtaaggacaa 720atggaacctc taaaattaaa atgaaatggg gaatggaaat gagacgctgc ctccttcagt 780ctctgcaaca gattgaaagc atgatcgaag ctgagtcctc agtcaaagaa aaggacatga 840ccaaagaatt ctttgagaac aaatcagaga catggcctat aggagagtcc cccaaaggag 900tggaagaggg ctcaatcggg aaggtttgca ggaccttatt agcaaaatct gtgtttaaca 960gtttgtatgc atctccacaa ctggaagggt tttcagctga atctaggaaa ttacttctca 1020ttgttcaggc ccttagggat aacctggaac ctggaacctt tgatattggg gggttatatg 1080aatcaattga ggagtgcctg attaatgatc cctgggtttt gctcaatgca tcttggttca 1140actccttcct tacacatgca ctgaagtagt tgtagcaatg ctactatttg ctatccatac 1200tgtccaaaaa agtactcgag ccc 1223 80 1233 DNA Equine influenza virus H3N8CDS (3)..(1172) 80 at gaa aag ggt ata aac cca aac tat ctc caa gct tggaag caa gta 47 Glu Lys Gly Ile Asn Pro Asn Tyr Leu Gln Ala Trp Lys GlnVal 1 5 10 15 tta gca gaa cta caa gac ctt gag aac gaa gaa aag acc cctaag acc 95 Leu Ala Glu Leu Gln Asp Leu Glu Asn Glu Glu Lys Thr Pro LysThr 20 25 30 aag aat atg aaa aaa aca agc caa ttg aaa tgg gca ctc ggt gaaaat 143 Lys Asn Met Lys Lys Thr Ser Gln Leu Lys Trp Ala Leu Gly Glu Asn35 40 45 atg gca cca gag aaa gtg gat ttt gag gat tgt aaa gac atc aat gat191 Met Ala Pro Glu Lys Val Asp Phe Glu Asp Cys Lys Asp Ile Asn Asp 5055 60 ttg aaa cag tat gac agt gat gag cca gaa aca agg tct ctt gca agt239 Leu Lys Gln Tyr Asp Ser Asp Glu Pro Glu Thr Arg Ser Leu Ala Ser 6570 75 tgg att caa agt gag ttc aac aaa gct tgt gag ctg aca gat tca agc287 Trp Ile Gln Ser Glu Phe Asn Lys Ala Cys Glu Leu Thr Asp Ser Ser 8085 90 95 tgg ata gag ctc gat gaa att ggg gag gat att gcc cca ata gaa tac335 Trp Ile Glu Leu Asp Glu Ile Gly Glu Asp Ile Ala Pro Ile Glu Tyr 100105 110 att gcg agc atg agg aga aat tat ttt act gct gag gtt tcc cat tgt383 Ile Ala Ser Met Arg Arg Asn Tyr Phe Thr Ala Glu Val Ser His Cys 115120 125 aga gca aca gaa tat ata atg aag gga gtg tac atc aac act gct cta431 Arg Ala Thr Glu Tyr Ile Met Lys Gly Val Tyr Ile Asn Thr Ala Leu 130135 140 ctc aat gca tcc tgt gct gcg atg gat gaa ttc caa tta att ccg atg479 Leu Asn Ala Ser Cys Ala Ala Met Asp Glu Phe Gln Leu Ile Pro Met 145150 155 ata agc aaa tgc agg acc aaa gaa ggg aga agg aag aca aat tta tat527 Ile Ser Lys Cys Arg Thr Lys Glu Gly Arg Arg Lys Thr Asn Leu Tyr 160165 170 175 gga ttc ata ata aag gga agg tcc cat tta agg aat gat acc gacgtg 575 Gly Phe Ile Ile Lys Gly Arg Ser His Leu Arg Asn Asp Thr Asp Val180 185 190 gta aac ttt gta agt atg gaa ttt tct ctc act gat cca aga tttgag 623 Val Asn Phe Val Ser Met Glu Phe Ser Leu Thr Asp Pro Arg Phe Glu195 200 205 cca cat aaa tgg gaa aaa tac tgc gtt cta gaa att gga gac atgctc 671 Pro His Lys Trp Glu Lys Tyr Cys Val Leu Glu Ile Gly Asp Met Leu210 215 220 cta agg act gct gta ggt caa gtg tca aga ccc atg ttt ttg tatgta 719 Leu Arg Thr Ala Val Gly Gln Val Ser Arg Pro Met Phe Leu Tyr Val225 230 235 agg aca aat gga acc tct aaa att aaa atg aaa tgg gga atg gaaatg 767 Arg Thr Asn Gly Thr Ser Lys Ile Lys Met Lys Trp Gly Met Glu Met240 245 250 255 aga cgc tgc ctc ctt cag tct ctg caa cag att gaa agc atgatc gaa 815 Arg Arg Cys Leu Leu Gln Ser Leu Gln Gln Ile Glu Ser Met IleGlu 260 265 270 gct gag tcc tca gtc aaa gaa aag gac atg acc aaa gaa ttcttt gag 863 Ala Glu Ser Ser Val Lys Glu Lys Asp Met Thr Lys Glu Phe PheGlu 275 280 285 aac aaa tca gag aca tgg cct ata gga gag tcc ccc aaa ggagtg gaa 911 Asn Lys Ser Glu Thr Trp Pro Ile Gly Glu Ser Pro Lys Gly ValGlu 290 295 300 gag ggc tca atc ggg aag gtt tgc agg acc tta tta gca aaatct gtg 959 Glu Gly Ser Ile Gly Lys Val Cys Arg Thr Leu Leu Ala Lys SerVal 305 310 315 ttt aac agt ttg tat gca tct cca caa ctg gaa ggg ttt tcagct gaa 1007 Phe Asn Ser Leu Tyr Ala Ser Pro Gln Leu Glu Gly Phe Ser AlaGlu 320 325 330 335 tct agg aaa tta ctt ctc att gtt cag gcc ctt agg gataac ctg gaa 1055 Ser Arg Lys Leu Leu Leu Ile Val Gln Ala Leu Arg Asp AsnLeu Glu 340 345 350 cct gga acc ttt gat att ggg ggg tta tat gaa tca attgag gag tgc 1103 Pro Gly Thr Phe Asp Ile Gly Gly Leu Tyr Glu Ser Ile GluGlu Cys 355 360 365 ctg att aat gat ccc tgg gtt ttg ctc aat gca tct tggttc aac tcc 1151 Leu Ile Asn Asp Pro Trp Val Leu Leu Asn Ala Ser Trp PheAsn Ser 370 375 380 ttc ctt aca cat gca ctg aag tagttgtagc aatgctactatttgctatcc 1202 Phe Leu Thr His Ala Leu Lys 385 390 atactgtccaaaaaagtacc ttgtttctac t 1233 81 390 PRT Equine influenza virus H3N8 81Glu Lys Gly Ile Asn Pro Asn Tyr Leu Gln Ala Trp Lys Gln Val Leu 1 5 1015 Ala Glu Leu Gln Asp Leu Glu Asn Glu Glu Lys Thr Pro Lys Thr Lys 20 2530 Asn Met Lys Lys Thr Ser Gln Leu Lys Trp Ala Leu Gly Glu Asn Met 35 4045 Ala Pro Glu Lys Val Asp Phe Glu Asp Cys Lys Asp Ile Asn Asp Leu 50 5560 Lys Gln Tyr Asp Ser Asp Glu Pro Glu Thr Arg Ser Leu Ala Ser Trp 65 7075 80 Ile Gln Ser Glu Phe Asn Lys Ala Cys Glu Leu Thr Asp Ser Ser Trp 8590 95 Ile Glu Leu Asp Glu Ile Gly Glu Asp Ile Ala Pro Ile Glu Tyr Ile100 105 110 Ala Ser Met Arg Arg Asn Tyr Phe Thr Ala Glu Val Ser His CysArg 115 120 125 Ala Thr Glu Tyr Ile Met Lys Gly Val Tyr Ile Asn Thr AlaLeu Leu 130 135 140 Asn Ala Ser Cys Ala Ala Met Asp Glu Phe Gln Leu IlePro Met Ile 145 150 155 160 Ser Lys Cys Arg Thr Lys Glu Gly Arg Arg LysThr Asn Leu Tyr Gly 165 170 175 Phe Ile Ile Lys Gly Arg Ser His Leu ArgAsn Asp Thr Asp Val Val 180 185 190 Asn Phe Val Ser Met Glu Phe Ser LeuThr Asp Pro Arg Phe Glu Pro 195 200 205 His Lys Trp Glu Lys Tyr Cys ValLeu Glu Ile Gly Asp Met Leu Leu 210 215 220 Arg Thr Ala Val Gly Gln ValSer Arg Pro Met Phe Leu Tyr Val Arg 225 230 235 240 Thr Asn Gly Thr SerLys Ile Lys Met Lys Trp Gly Met Glu Met Arg 245 250 255 Arg Cys Leu LeuGln Ser Leu Gln Gln Ile Glu Ser Met Ile Glu Ala 260 265 270 Glu Ser SerVal Lys Glu Lys Asp Met Thr Lys Glu Phe Phe Glu Asn 275 280 285 Lys SerGlu Thr Trp Pro Ile Gly Glu Ser Pro Lys Gly Val Glu Glu 290 295 300 GlySer Ile Gly Lys Val Cys Arg Thr Leu Leu Ala Lys Ser Val Phe 305 310 315320 Asn Ser Leu Tyr Ala Ser Pro Gln Leu Glu Gly Phe Ser Ala Glu Ser 325330 335 Arg Lys Leu Leu Leu Ile Val Gln Ala Leu Arg Asp Asn Leu Glu Pro340 345 350 Gly Thr Phe Asp Ile Gly Gly Leu Tyr Glu Ser Ile Glu Glu CysLeu 355 360 365 Ile Asn Asp Pro Trp Val Leu Leu Asn Ala Ser Trp Phe AsnSer Phe 370 375 380 Leu Thr His Ala Leu Lys 385 390 82 1170 DNA Equineinfluenza virus H3N8 82 gaaaagggta taaacccaaa ctatctccaa gcttggaagcaagtattagc agaactacaa 60 gaccttgaga acgaagaaaa gacccctaag accaagaatatgaaaaaaac aagccaattg 120 aaatgggcac tcggtgaaaa tatggcacca gagaaagtggattttgagga ttgtaaagac 180 atcaatgatt tgaaacagta tgacagtgat gagccagaaacaaggtctct tgcaagttgg 240 attcaaagtg agttcaacaa agcttgtgag ctgacagattcaagctggat agagctcgat 300 gaaattgggg aggatattgc cccaatagaa tacattgcgagcatgaggag aaattatttt 360 actgctgagg tttcccattg tagagcaaca gaatatataatgaagggagt gtacatcaac 420 actgctctac tcaatgcatc ctgtgctgcg atggatgaattccaattaat tccgatgata 480 agcaaatgca ggaccaaaga agggagaagg aagacaaatttatatggatt cataataaag 540 ggaaggtccc atttaaggaa tgataccgac gtggtaaactttgtaagtat ggaattttct 600 ctcactgatc caagatttga gccacataaa tgggaaaaatactgcgttct agaaattgga 660 gacatgctcc taaggactgc tgtaggtcaa gtgtcaagacccatgttttt gtatgtaagg 720 acaaatggaa cctctaaaat taaaatgaaa tggggaatggaaatgagacg ctgcctcctt 780 cagtctctgc aacagattga aagcatgatc gaagctgagtcctcagtcaa agaaaaggac 840 atgaccaaag aattctttga gaacaaatca gagacatggcctataggaga gtcccccaaa 900 ggagtggaag agggctcaat cgggaaggtt tgcaggaccttattagcaaa atctgtgttt 960 aacagtttgt atgcatctcc acaactggaa gggttttcagctgaatctag gaaattactt 1020 ctcattgttc aggcccttag ggataacctg gaacctggaacctttgatat tggggggtta 1080 tatgaatcaa ttgaggagtg cctgattaat gatccctgggttttgctcaa tgcatcttgg 1140 ttcaactcct tccttacaca tgcactgaag 1170 83 25DNA Artificial Sequence Description of Artificial Sequence SyntheticPrimer 83 ggggcgggta cccaaactat ctcca 25 84 27 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Primer 84 gggggctcgagtactttttt ggacagt 27 85 1234 DNA Equine influenza virus H3N8 CDS(1)..(1188) 85 atg aaa ttg aga act caa ata cca gca gaa atg ctc gca agcatt gat 48 Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met Leu Ala Ser IleAsp 1 5 10 15 ctg aaa tat ttc aat gat tca aca aaa aag aaa att gag aagata cga 96 Leu Lys Tyr Phe Asn Asp Ser Thr Lys Lys Lys Ile Glu Lys IleArg 20 25 30 cca ctt ctg gtc gat ggg act gct tca ctg agt cct ggc atg atgatg 144 Pro Leu Leu Val Asp Gly Thr Ala Ser Leu Ser Pro Gly Met Met Met35 40 45 gga atg ttc aac atg ttg agc act gta cta ggt gta tcc ata tta aac192 Gly Met Phe Asn Met Leu Ser Thr Val Leu Gly Val Ser Ile Leu Asn 5055 60 ctg ggc cag agg aaa tac aca aag acc aca tac tgg tgg gat ggt ctg240 Leu Gly Gln Arg Lys Tyr Thr Lys Thr Thr Tyr Trp Trp Asp Gly Leu 6570 75 80 caa tca tcc gat gat ttt gct ttg ata gtg aat gcg cct aat cat gaa288 Gln Ser Ser Asp Asp Phe Ala Leu Ile Val Asn Ala Pro Asn His Glu 8590 95 gga ata cag gct gga gta gac aga ttc tat aga act tgc aaa ctg gtc336 Gly Ile Gln Ala Gly Val Asp Arg Phe Tyr Arg Thr Cys Lys Leu Val 100105 110 ggg atc aac atg agc aaa aag aag tcc tac ata aat aga acc ggc aca384 Gly Ile Asn Met Ser Lys Lys Lys Ser Tyr Ile Asn Arg Thr Gly Thr 115120 125 ttc gaa ttc aca agc ttt ttc tac cgg tat ggt ttt gtc gcc aat ttc432 Phe Glu Phe Thr Ser Phe Phe Tyr Arg Tyr Gly Phe Val Ala Asn Phe 130135 140 agc atg gag cta ccc agt ttt ggg gtt tcc ggg ata aat gaa tct gca480 Ser Met Glu Leu Pro Ser Phe Gly Val Ser Gly Ile Asn Glu Ser Ala 145150 155 160 gac atg agc att gga atg aca gtt atc aaa aac aac atg ata aataat 528 Asp Met Ser Ile Gly Met Thr Val Ile Lys Asn Asn Met Ile Asn Asn165 170 175 gat ctc ggt ccc gcc acg gca caa atg gca ctc caa ctc ttc attaag 576 Asp Leu Gly Pro Ala Thr Ala Gln Met Ala Leu Gln Leu Phe Ile Lys180 185 190 gat tat cgg tac aca tac cgg tgc cat aga ggc gat acc cag atacaa 624 Asp Tyr Arg Tyr Thr Tyr Arg Cys His Arg Gly Asp Thr Gln Ile Gln195 200 205 acc aga aga tcc ttt gag ttg aag aaa ctg tgg gaa cag act cgatca 672 Thr Arg Arg Ser Phe Glu Leu Lys Lys Leu Trp Glu Gln Thr Arg Ser210 215 220 aag act ggt cta ctg gta tca gat ggg ggt cca aac cta tac aacatc 720 Lys Thr Gly Leu Leu Val Ser Asp Gly Gly Pro Asn Leu Tyr Asn Ile225 230 235 240 aga aac cta cac atc ccg gaa gtc tgt ttg aaa tgg gag ctgatg gat 768 Arg Asn Leu His Ile Pro Glu Val Cys Leu Lys Trp Glu Leu MetAsp 245 250 255 gaa gat tat aaa ggg agg cta tgt aat cca ttg aat cct ttcgtt agc 816 Glu Asp Tyr Lys Gly Arg Leu Cys Asn Pro Leu Asn Pro Phe ValSer 260 265 270 cac aaa gaa att gaa tca gtg aac agt gca gta gta atg cctgcg cat 864 His Lys Glu Ile Glu Ser Val Asn Ser Ala Val Val Met Pro AlaHis 275 280 285 ggc cct gcc aaa agc atg gag tat gat gct gtt gca aca acacac tct 912 Gly Pro Ala Lys Ser Met Glu Tyr Asp Ala Val Ala Thr Thr HisSer 290 295 300 tgg atc ccc aag agg aac cgg tcc ata ttg aac aca agt caaagg gga 960 Trp Ile Pro Lys Arg Asn Arg Ser Ile Leu Asn Thr Ser Gln ArgGly 305 310 315 320 ata ctc gaa gat gag cag atg tat cag aaa tgc tgc aacctg ttt gaa 1008 Ile Leu Glu Asp Glu Gln Met Tyr Gln Lys Cys Cys Asn LeuPhe Glu 325 330 335 aaa ttc ttc ccc agc agc tca tac aga aga cca gtc ggaatt tct agt 1056 Lys Phe Phe Pro Ser Ser Ser Tyr Arg Arg Pro Val Gly IleSer Ser 340 345 350 atg gtt gag gcc atg gtg tcc agg gcc cgc att gat gcacga att gac 1104 Met Val Glu Ala Met Val Ser Arg Ala Arg Ile Asp Ala ArgIle Asp 355 360 365 ttc gaa tct gga cgg ata aag aag gat gag ttc gct gagatc atg aag 1152 Phe Glu Ser Gly Arg Ile Lys Lys Asp Glu Phe Ala Glu IleMet Lys 370 375 380 atc tgt tcc acc att gaa gag ctc aga cgg caa aaatagtgaattt 1198 Ile Cys Ser Thr Ile Glu Glu Leu Arg Arg Gln Lys 385 390395 agcttgatct tcgtgaaaaa atgccttgtt tctact 1234 86 396 PRT Equineinfluenza virus H3N8 86 Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met LeuAla Ser Ile Asp 1 5 10 15 Leu Lys Tyr Phe Asn Asp Ser Thr Lys Lys LysIle Glu Lys Ile Arg 20 25 30 Pro Leu Leu Val Asp Gly Thr Ala Ser Leu SerPro Gly Met Met Met 35 40 45 Gly Met Phe Asn Met Leu Ser Thr Val Leu GlyVal Ser Ile Leu Asn 50 55 60 Leu Gly Gln Arg Lys Tyr Thr Lys Thr Thr TyrTrp Trp Asp Gly Leu 65 70 75 80 Gln Ser Ser Asp Asp Phe Ala Leu Ile ValAsn Ala Pro Asn His Glu 85 90 95 Gly Ile Gln Ala Gly Val Asp Arg Phe TyrArg Thr Cys Lys Leu Val 100 105 110 Gly Ile Asn Met Ser Lys Lys Lys SerTyr Ile Asn Arg Thr Gly Thr 115 120 125 Phe Glu Phe Thr Ser Phe Phe TyrArg Tyr Gly Phe Val Ala Asn Phe 130 135 140 Ser Met Glu Leu Pro Ser PheGly Val Ser Gly Ile Asn Glu Ser Ala 145 150 155 160 Asp Met Ser Ile GlyMet Thr Val Ile Lys Asn Asn Met Ile Asn Asn 165 170 175 Asp Leu Gly ProAla Thr Ala Gln Met Ala Leu Gln Leu Phe Ile Lys 180 185 190 Asp Tyr ArgTyr Thr Tyr Arg Cys His Arg Gly Asp Thr Gln Ile Gln 195 200 205 Thr ArgArg Ser Phe Glu Leu Lys Lys Leu Trp Glu Gln Thr Arg Ser 210 215 220 LysThr Gly Leu Leu Val Ser Asp Gly Gly Pro Asn Leu Tyr Asn Ile 225 230 235240 Arg Asn Leu His Ile Pro Glu Val Cys Leu Lys Trp Glu Leu Met Asp 245250 255 Glu Asp Tyr Lys Gly Arg Leu Cys Asn Pro Leu Asn Pro Phe Val Ser260 265 270 His Lys Glu Ile Glu Ser Val Asn Ser Ala Val Val Met Pro AlaHis 275 280 285 Gly Pro Ala Lys Ser Met Glu Tyr Asp Ala Val Ala Thr ThrHis Ser 290 295 300 Trp Ile Pro Lys Arg Asn Arg Ser Ile Leu Asn Thr SerGln Arg Gly 305 310 315 320 Ile Leu Glu Asp Glu Gln Met Tyr Gln Lys CysCys Asn Leu Phe Glu 325 330 335 Lys Phe Phe Pro Ser Ser Ser Tyr Arg ArgPro Val Gly Ile Ser Ser 340 345 350 Met Val Glu Ala Met Val Ser Arg AlaArg Ile Asp Ala Arg Ile Asp 355 360 365 Phe Glu Ser Gly Arg Ile Lys LysAsp Glu Phe Ala Glu Ile Met Lys 370 375 380 Ile Cys Ser Thr Ile Glu GluLeu Arg Arg Gln Lys 385 390 395 87 1188 DNA Equine influenza virus H3N887 atgaaattga gaactcaaat accagcagaa atgctcgcaa gcattgatct gaaatatttc 60aatgattcaa caaaaaagaa aattgagaag atacgaccac ttctggtcga tgggactgct 120tcactgagtc ctggcatgat gatgggaatg ttcaacatgt tgagcactgt actaggtgta 180tccatattaa acctgggcca gaggaaatac acaaagacca catactggtg ggatggtctg 240caatcatccg atgattttgc tttgatagtg aatgcgccta atcatgaagg aatacaggct 300ggagtagaca gattctatag aacttgcaaa ctggtcggga tcaacatgag caaaaagaag 360tcctacataa atagaaccgg cacattcgaa ttcacaagct ttttctaccg gtatggtttt 420gtcgccaatt tcagcatgga gctacccagt tttggggttt ccgggataaa tgaatctgca 480gacatgagca ttggaatgac agttatcaaa aacaacatga taaataatga tctcggtccc 540gccacggcac aaatggcact ccaactcttc attaaggatt atcggtacac ataccggtgc 600catagaggcg atacccagat acaaaccaga agatcctttg agttgaagaa actgtgggaa 660cagactcgat caaagactgg tctactggta tcagatgggg gtccaaacct atacaacatc 720agaaacctac acatcccgga agtctgtttg aaatgggagc tgatggatga agattataaa 780gggaggctat gtaatccatt gaatcctttc gttagccaca aagaaattga atcagtgaac 840agtgcagtag taatgcctgc gcatggccct gccaaaagca tggagtatga tgctgttgca 900acaacacact cttggatccc caagaggaac cggtccatat tgaacacaag tcaaagggga 960atactcgaag atgagcagat gtatcagaaa tgctgcaacc tgtttgaaaa attcttcccc 1020agcagctcat acagaagacc agtcggaatt tctagtatgg ttgaggccat ggtgtccagg 1080gcccgcattg atgcacgaat tgacttcgaa tctggacgga taaagaagga tgagttcgct 1140gagatcatga agatctgttc caccattgaa gagctcagac ggcaaaaa 1188 88 1240 DNAEquine influenza virus H3N8 CDS (8)..(1195) 88 caaaagt atg aaa ttg agaact caa ata cca gca gaa atg ctc gca agc 49 Met Lys Leu Arg Thr Gln IlePro Ala Glu Met Leu Ala Ser 1 5 10 att gat ctg aaa tat ttc aat gat tcaaca aaa aag aaa att gag aag 97 Ile Asp Leu Lys Tyr Phe Asn Asp Ser ThrLys Lys Lys Ile Glu Lys 15 20 25 30 ata cga cca ctt ctg gtc gat ggg actgct tca ctg agt cct ggc atg 145 Ile Arg Pro Leu Leu Val Asp Gly Thr AlaSer Leu Ser Pro Gly Met 35 40 45 atg atg gga atg ttc aac atg ttg agc actgta cta ggt gta tcc ata 193 Met Met Gly Met Phe Asn Met Leu Ser Thr ValLeu Gly Val Ser Ile 50 55 60 tta aac ctg ggc cag agg aaa tac aca aag accaca tac tgg tgg gat 241 Leu Asn Leu Gly Gln Arg Lys Tyr Thr Lys Thr ThrTyr Trp Trp Asp 65 70 75 ggt ctg caa tca tcc gat gat ttt gct ttg ata gtgaat gcg cct aat 289 Gly Leu Gln Ser Ser Asp Asp Phe Ala Leu Ile Val AsnAla Pro Asn 80 85 90 cat gaa gga ata cag gct gga gta gac aga ttc tat agaact tgc aaa 337 His Glu Gly Ile Gln Ala Gly Val Asp Arg Phe Tyr Arg ThrCys Lys 95 100 105 110 ctg gtc ggg atc aac atg agc aaa aag aag tcc tacata aat aga acc 385 Leu Val Gly Ile Asn Met Ser Lys Lys Lys Ser Tyr IleAsn Arg Thr 115 120 125 ggc tca ttc gaa ttc aca agc ttt ttc tac cgg tatggt ttt gtc gcc 433 Gly Ser Phe Glu Phe Thr Ser Phe Phe Tyr Arg Tyr GlyPhe Val Ala 130 135 140 aat ttc agc atg gag cta ccc agt ttt ggg gtt tccggg ata aat gaa 481 Asn Phe Ser Met Glu Leu Pro Ser Phe Gly Val Ser GlyIle Asn Glu 145 150 155 tct gca gac atg agc att gga atg aca gtt atc aaaaac aac atg ata 529 Ser Ala Asp Met Ser Ile Gly Met Thr Val Ile Lys AsnAsn Met Ile 160 165 170 aat aat gat ctc ggt ccc gcc acg gca caa atg gcactc caa ctc ttc 577 Asn Asn Asp Leu Gly Pro Ala Thr Ala Gln Met Ala LeuGln Leu Phe 175 180 185 190 att aag gat tat cgg tac aca tac cgg tgc cataga ggc gat acc cag 625 Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg Cys His ArgGly Asp Thr Gln 195 200 205 ata caa acc aga aga tcc ttt gag ttg aag aaactg tgg gaa cag act 673 Ile Gln Thr Arg Arg Ser Phe Glu Leu Lys Lys LeuTrp Glu Gln Thr 210 215 220 cga tca aag act ggt cta ctg gta tca gat gggggt cca aac cta tac 721 Arg Ser Lys Thr Gly Leu Leu Val Ser Asp Gly GlyPro Asn Leu Tyr 225 230 235 aac atc aga aac cta cac atc ccg gaa gtc tgtttg aaa tgg gag ctg 769 Asn Ile Arg Asn Leu His Ile Pro Glu Val Cys LeuLys Trp Glu Leu 240 245 250 atg gat gaa gat tat aaa ggg agg cta tgt aatcca ttg aat cct ttc 817 Met Asp Glu Asp Tyr Lys Gly Arg Leu Cys Asn ProLeu Asn Pro Phe 255 260 265 270 gtt agc cac aaa gaa att gaa tca gtg aacagt gca gta gta atg cct 865 Val Ser His Lys Glu Ile Glu Ser Val Asn SerAla Val Val Met Pro 275 280 285 gcg cat ggc cct gcc aaa agc atg gag tatgat gct gtt gca aca aca 913 Ala His Gly Pro Ala Lys Ser Met Glu Tyr AspAla Val Ala Thr Thr 290 295 300 cac tct tgg atc ccc aag agg aac cgg tccata ttg aac aca agt caa 961 His Ser Trp Ile Pro Lys Arg Asn Arg Ser IleLeu Asn Thr Ser Gln 305 310 315 agg gga ata ctc gaa gat gag cag atg tatcag aaa tgc tgc aac ctg 1009 Arg Gly Ile Leu Glu Asp Glu Gln Met Tyr GlnLys Cys Cys Asn Leu 320 325 330 ttt gaa aaa ttc ttc ccc agc agc tca tacaga aga cca gtc gga att 1057 Phe Glu Lys Phe Phe Pro Ser Ser Ser Tyr ArgArg Pro Val Gly Ile 335 340 345 350 tct agt atg gtt gag gcc atg gtg tccagg gcc cgc att gat gca cga 1105 Ser Ser Met Val Glu Ala Met Val Ser ArgAla Arg Ile Asp Ala Arg 355 360 365 att gac ttc gaa tct gga cgg ata aagaag gat gag ttc gct gag atc 1153 Ile Asp Phe Glu Ser Gly Arg Ile Lys LysAsp Glu Phe Ala Glu Ile 370 375 380 atg aag atc tgt tcc acc att gaa gagctc aga cgg caa aaa 1195 Met Lys Ile Cys Ser Thr Ile Glu Glu Leu Arg ArgGln Lys 385 390 395 tagtgaattt agcttgatct tcgtgaaaaa atgccttgtt ctact1240 89 396 PRT Equine influenza virus H3N8 89 Met Lys Leu Arg Thr GlnIle Pro Ala Glu Met Leu Ala Ser Ile Asp 1 5 10 15 Leu Lys Tyr Phe AsnAsp Ser Thr Lys Lys Lys Ile Glu Lys Ile Arg 20 25 30 Pro Leu Leu Val AspGly Thr Ala Ser Leu Ser Pro Gly Met Met Met 35 40 45 Gly Met Phe Asn MetLeu Ser Thr Val Leu Gly Val Ser Ile Leu Asn 50 55 60 Leu Gly Gln Arg LysTyr Thr Lys Thr Thr Tyr Trp Trp Asp Gly Leu 65 70 75 80 Gln Ser Ser AspAsp Phe Ala Leu Ile Val Asn Ala Pro Asn His Glu 85 90 95 Gly Ile Gln AlaGly Val Asp Arg Phe Tyr Arg Thr Cys Lys Leu Val 100 105 110 Gly Ile AsnMet Ser Lys Lys Lys Ser Tyr Ile Asn Arg Thr Gly Ser 115 120 125 Phe GluPhe Thr Ser Phe Phe Tyr Arg Tyr Gly Phe Val Ala Asn Phe 130 135 140 SerMet Glu Leu Pro Ser Phe Gly Val Ser Gly Ile Asn Glu Ser Ala 145 150 155160 Asp Met Ser Ile Gly Met Thr Val Ile Lys Asn Asn Met Ile Asn Asn 165170 175 Asp Leu Gly Pro Ala Thr Ala Gln Met Ala Leu Gln Leu Phe Ile Lys180 185 190 Asp Tyr Arg Tyr Thr Tyr Arg Cys His Arg Gly Asp Thr Gln IleGln 195 200 205 Thr Arg Arg Ser Phe Glu Leu Lys Lys Leu Trp Glu Gln ThrArg Ser 210 215 220 Lys Thr Gly Leu Leu Val Ser Asp Gly Gly Pro Asn LeuTyr Asn Ile 225 230 235 240 Arg Asn Leu His Ile Pro Glu Val Cys Leu LysTrp Glu Leu Met Asp 245 250 255 Glu Asp Tyr Lys Gly Arg Leu Cys Asn ProLeu Asn Pro Phe Val Ser 260 265 270 His Lys Glu Ile Glu Ser Val Asn SerAla Val Val Met Pro Ala His 275 280 285 Gly Pro Ala Lys Ser Met Glu TyrAsp Ala Val Ala Thr Thr His Ser 290 295 300 Trp Ile Pro Lys Arg Asn ArgSer Ile Leu Asn Thr Ser Gln Arg Gly 305 310 315 320 Ile Leu Glu Asp GluGln Met Tyr Gln Lys Cys Cys Asn Leu Phe Glu 325 330 335 Lys Phe Phe ProSer Ser Ser Tyr Arg Arg Pro Val Gly Ile Ser Ser 340 345 350 Met Val GluAla Met Val Ser Arg Ala Arg Ile Asp Ala Arg Ile Asp 355 360 365 Phe GluSer Gly Arg Ile Lys Lys Asp Glu Phe Ala Glu Ile Met Lys 370 375 380 IleCys Ser Thr Ile Glu Glu Leu Arg Arg Gln Lys 385 390 395 90 1188 DNAEquine influenza virus H3N8 90 atgaaattga gaactcaaat accagcagaaatgctcgcaa gcattgatct gaaatatttc 60 aatgattcaa caaaaaagaa aattgagaagatacgaccac ttctggtcga tgggactgct 120 tcactgagtc ctggcatgat gatgggaatgttcaacatgt tgagcactgt actaggtgta 180 tccatattaa acctgggcca gaggaaatacacaaagacca catactggtg ggatggtctg 240 caatcatccg atgattttgc tttgatagtgaatgcgccta atcatgaagg aatacaggct 300 ggagtagaca gattctatag aacttgcaaactggtcggga tcaacatgag caaaaagaag 360 tcctacataa atagaaccgg ctcattcgaattcacaagct ttttctaccg gtatggtttt 420 gtcgccaatt tcagcatgga gctacccagttttggggttt ccgggataaa tgaatctgca 480 gacatgagca ttggaatgac agttatcaaaaacaacatga taaataatga tctcggtccc 540 gccacggcac aaatggcact ccaactcttcattaaggatt atcggtacac ataccggtgc 600 catagaggcg atacccagat acaaaccagaagatcctttg agttgaagaa actgtgggaa 660 cagactcgat caaagactgg tctactggtatcagatgggg gtccaaacct atacaacatc 720 agaaacctac acatcccgga agtctgtttgaaatgggagc tgatggatga agattataaa 780 gggaggctat gtaatccatt gaatcctttcgttagccaca aagaaattga atcagtgaac 840 agtgcagtag taatgcctgc gcatggccctgccaaaagca tggagtatga tgctgttgca 900 acaacacact cttggatccc caagaggaaccggtccatat tgaacacaag tcaaagggga 960 atactcgaag atgagcagat gtatcagaaatgctgcaacc tgtttgaaaa attcttcccc 1020 agcagctcat acagaagacc agtcggaatttctagtatgg ttgaggccat ggtgtccagg 1080 gcccgcattg atgcacgaat tgacttcgaatctggacgga taaagaagga tgagttcgct 1140 gagatcatga agatctgttc caccattgaagagctcagac ggcaaaaa 1188 91 1241 DNA Equine influenza virus H3N8 CDS(8)..(1195) 91 caaaagt atg aaa ttg aga act caa ata cca gca gaa atg ctcgca agc 49 Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met Leu Ala Ser 1 510 att gat ctg aaa tat ttc aat gat tca aca aaa aag aaa att gag aag 97Ile Asp Leu Lys Tyr Phe Asn Asp Ser Thr Lys Lys Lys Ile Glu Lys 15 20 2530 ata cga cca ctt ctg gtc gat ggg act gct tca ctg agt cct ggc atg 145Ile Arg Pro Leu Leu Val Asp Gly Thr Ala Ser Leu Ser Pro Gly Met 35 40 45atg atg gga atg ttc aac atg ttg agc act gta cta ggt gta tcc ata 193 MetMet Gly Met Phe Asn Met Leu Ser Thr Val Leu Gly Val Ser Ile 50 55 60 ttaaac ctg ggc cag agg aaa tac aca aag acc aca tac tgg tgg gat 241 Leu AsnLeu Gly Gln Arg Lys Tyr Thr Lys Thr Thr Tyr Trp Trp Asp 65 70 75 ggt ctgcaa tca tcc gat gat ttt gct ttg ata gtg aat gcg cct aat 289 Gly Leu GlnSer Ser Asp Asp Phe Ala Leu Ile Val Asn Ala Pro Asn 80 85 90 cat gaa ggaata cag gct gga gta gac aga ttc tat aga act tgc aaa 337 His Glu Gly IleGln Ala Gly Val Asp Arg Phe Tyr Arg Thr Cys Lys 95 100 105 110 ctg gtcggg atc aac atg agc aaa aag aag tcc tac ata aat aga acc 385 Leu Val GlyIle Asn Met Ser Lys Lys Lys Ser Tyr Ile Asn Arg Thr 115 120 125 ggc acattc gaa ttc aca agc ttt ttc tac cgg tat ggt ttt gtc gcc 433 Gly Thr PheGlu Phe Thr Ser Phe Phe Tyr Arg Tyr Gly Phe Val Ala 130 135 140 aat ttcagc atg gag cta ccc agt ttt ggg gtt tcc ggg ata aat gaa 481 Asn Phe SerMet Glu Leu Pro Ser Phe Gly Val Ser Gly Ile Asn Glu 145 150 155 tct gcagac atg agc att gga atg aca gtt atc aaa aac aac atg ata 529 Ser Ala AspMet Ser Ile Gly Met Thr Val Ile Lys Asn Asn Met Ile 160 165 170 aat aatgat ctc ggt ccc gcc acg gca caa atg gca ctc caa ctc ttc 577 Asn Asn AspLeu Gly Pro Ala Thr Ala Gln Met Ala Leu Gln Leu Phe 175 180 185 190 attaag gat tat cgg tac aca tac cgg tgt caa aga ggc gat acc cag 625 Ile LysAsp Tyr Arg Tyr Thr Tyr Arg Cys Gln Arg Gly Asp Thr Gln 195 200 205 atacaa acc aga aga tcc ttt gag ttg aag aaa ctg tgg gaa cag act 673 Ile GlnThr Arg Arg Ser Phe Glu Leu Lys Lys Leu Trp Glu Gln Thr 210 215 220 cgatca aag act ggt cta ctg gta tca gat ggg ggt cca aac cta tac 721 Arg SerLys Thr Gly Leu Leu Val Ser Asp Gly Gly Pro Asn Leu Tyr 225 230 235 aacatc aga aac cta cac atc ccg gaa gtc tgt ttg aaa tgg gag ctg 769 Asn IleArg Asn Leu His Ile Pro Glu Val Cys Leu Lys Trp Glu Leu 240 245 250 atggat gaa gat tat aaa ggg agg cta tgt aat cca ttg aat cct ttc 817 Met AspGlu Asp Tyr Lys Gly Arg Leu Cys Asn Pro Leu Asn Pro Phe 255 260 265 270gtt agc cac aaa gaa att gaa tca gtg aac agt gca gta gta atg cct 865 ValSer His Lys Glu Ile Glu Ser Val Asn Ser Ala Val Val Met Pro 275 280 285gcg cat ggc cct gcc aaa agc atg gag tat gat gct gtt gca aca aca 913 AlaHis Gly Pro Ala Lys Ser Met Glu Tyr Asp Ala Val Ala Thr Thr 290 295 300cac tct tgg atc ccc aag agg aac cgg tcc ata ttg aac aca agt caa 961 HisSer Trp Ile Pro Lys Arg Asn Arg Ser Ile Leu Asn Thr Ser Gln 305 310 315agg gga ata ctc gaa gat gag cag atg tat cag aaa tgc tgc aac ctg 1009 ArgGly Ile Leu Glu Asp Glu Gln Met Tyr Gln Lys Cys Cys Asn Leu 320 325 330ttt gaa aaa ttc ttc ccc agc agc tca tac aga aaa cca gtc gga att 1057 PheGlu Lys Phe Phe Pro Ser Ser Ser Tyr Arg Lys Pro Val Gly Ile 335 340 345350 tct agt atg gtt gag gcc atg gtg tcc agg gcc cgc att gat gca cga 1105Ser Ser Met Val Glu Ala Met Val Ser Arg Ala Arg Ile Asp Ala Arg 355 360365 att gac ttc gaa tct gga cgg ata aag aag gat gag ttc gct gag atc 1153Ile Asp Phe Glu Ser Gly Arg Ile Lys Lys Asp Glu Phe Ala Glu Ile 370 375380 atg aag atc tgt tcc acc att gaa gag ctc aga cgg caa aaa 1195 Met LysIle Cys Ser Thr Ile Glu Glu Leu Arg Arg Gln Lys 385 390 395 tagtgaatttagcttgatct tcgtgaaaaa atgccttgtt tctact 1241 92 396 PRT Equine influenzavirus H3N8 92 Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met Leu Ala SerIle Asp 1 5 10 15 Leu Lys Tyr Phe Asn Asp Ser Thr Lys Lys Lys Ile GluLys Ile Arg 20 25 30 Pro Leu Leu Val Asp Gly Thr Ala Ser Leu Ser Pro GlyMet Met Met 35 40 45 Gly Met Phe Asn Met Leu Ser Thr Val Leu Gly Val SerIle Leu Asn 50 55 60 Leu Gly Gln Arg Lys Tyr Thr Lys Thr Thr Tyr Trp TrpAsp Gly Leu 65 70 75 80 Gln Ser Ser Asp Asp Phe Ala Leu Ile Val Asn AlaPro Asn His Glu 85 90 95 Gly Ile Gln Ala Gly Val Asp Arg Phe Tyr Arg ThrCys Lys Leu Val 100 105 110 Gly Ile Asn Met Ser Lys Lys Lys Ser Tyr IleAsn Arg Thr Gly Thr 115 120 125 Phe Glu Phe Thr Ser Phe Phe Tyr Arg TyrGly Phe Val Ala Asn Phe 130 135 140 Ser Met Glu Leu Pro Ser Phe Gly ValSer Gly Ile Asn Glu Ser Ala 145 150 155 160 Asp Met Ser Ile Gly Met ThrVal Ile Lys Asn Asn Met Ile Asn Asn 165 170 175 Asp Leu Gly Pro Ala ThrAla Gln Met Ala Leu Gln Leu Phe Ile Lys 180 185 190 Asp Tyr Arg Tyr ThrTyr Arg Cys Gln Arg Gly Asp Thr Gln Ile Gln 195 200 205 Thr Arg Arg SerPhe Glu Leu Lys Lys Leu Trp Glu Gln Thr Arg Ser 210 215 220 Lys Thr GlyLeu Leu Val Ser Asp Gly Gly Pro Asn Leu Tyr Asn Ile 225 230 235 240 ArgAsn Leu His Ile Pro Glu Val Cys Leu Lys Trp Glu Leu Met Asp 245 250 255Glu Asp Tyr Lys Gly Arg Leu Cys Asn Pro Leu Asn Pro Phe Val Ser 260 265270 His Lys Glu Ile Glu Ser Val Asn Ser Ala Val Val Met Pro Ala His 275280 285 Gly Pro Ala Lys Ser Met Glu Tyr Asp Ala Val Ala Thr Thr His Ser290 295 300 Trp Ile Pro Lys Arg Asn Arg Ser Ile Leu Asn Thr Ser Gln ArgGly 305 310 315 320 Ile Leu Glu Asp Glu Gln Met Tyr Gln Lys Cys Cys AsnLeu Phe Glu 325 330 335 Lys Phe Phe Pro Ser Ser Ser Tyr Arg Lys Pro ValGly Ile Ser Ser 340 345 350 Met Val Glu Ala Met Val Ser Arg Ala Arg IleAsp Ala Arg Ile Asp 355 360 365 Phe Glu Ser Gly Arg Ile Lys Lys Asp GluPhe Ala Glu Ile Met Lys 370 375 380 Ile Cys Ser Thr Ile Glu Glu Leu ArgArg Gln Lys 385 390 395 93 1188 DNA Equine influenza virus H3N8 93atgaaattga gaactcaaat accagcagaa atgctcgcaa gcattgatct gaaatatttc 60aatgattcaa caaaaaagaa aattgagaag atacgaccac ttctggtcga tgggactgct 120tcactgagtc ctggcatgat gatgggaatg ttcaacatgt tgagcactgt actaggtgta 180tccatattaa acctgggcca gaggaaatac acaaagacca catactggtg ggatggtctg 240caatcatccg atgattttgc tttgatagtg aatgcgccta atcatgaagg aatacaggct 300ggagtagaca gattctatag aacttgcaaa ctggtcggga tcaacatgag caaaaagaag 360tcctacataa atagaaccgg cacattcgaa ttcacaagct ttttctaccg gtatggtttt 420gtcgccaatt tcagcatgga gctacccagt tttggggttt ccgggataaa tgaatctgca 480gacatgagca ttggaatgac agttatcaaa aacaacatga taaataatga tctcggtccc 540gccacggcac aaatggcact ccaactcttc attaaggatt atcggtacac ataccggtgt 600caaagaggcg atacccagat acaaaccaga agatcctttg agttgaagaa actgtgggaa 660cagactcgat caaagactgg tctactggta tcagatgggg gtccaaacct atacaacatc 720agaaacctac acatcccgga agtctgtttg aaatgggagc tgatggatga agattataaa 780gggaggctat gtaatccatt gaatcctttc gttagccaca aagaaattga atcagtgaac 840agtgcagtag taatgcctgc gcatggccct gccaaaagca tggagtatga tgctgttgca 900acaacacact cttggatccc caagaggaac cggtccatat tgaacacaag tcaaagggga 960atactcgaag atgagcagat gtatcagaaa tgctgcaacc tgtttgaaaa attcttcccc 1020agcagctcat acagaaaacc agtcggaatt tctagtatgg ttgaggccat ggtgtccagg 1080gcccgcattg atgcacgaat tgacttcgaa tctggacgga taaagaagga tgagttcgct 1140gagatcatga agatctgttc caccattgaa gagctcagac ggcaaaaa 1188 94 1241 DNAEquine influenza virus H3N8 CDS (8)..(1195) 94 caaaagt atg aaa ttg agaact caa ata cca gca gaa atg ctc gca agc 49 Met Lys Leu Arg Thr Gln IlePro Ala Glu Met Leu Ala Ser 1 5 10 att gat ctg aaa tat ttc aat gat tcaaca aaa aag aaa att gag aag 97 Ile Asp Leu Lys Tyr Phe Asn Asp Ser ThrLys Lys Lys Ile Glu Lys 15 20 25 30 ata cga cca ctt ctg gtc gat ggg actgct tca ctg agt cct ggc atg 145 Ile Arg Pro Leu Leu Val Asp Gly Thr AlaSer Leu Ser Pro Gly Met 35 40 45 atg atg gga atg ttc aac atg ttg agc actgta cta ggt gta tcc ata 193 Met Met Gly Met Phe Asn Met Leu Ser Thr ValLeu Gly Val Ser Ile 50 55 60 tta aac ctg ggc cag agg aaa tac aca aag accaca tac tgg tgg gat 241 Leu Asn Leu Gly Gln Arg Lys Tyr Thr Lys Thr ThrTyr Trp Trp Asp 65 70 75 ggt ctg caa tca tcc gat gat ttt gct ttg ata gtgaat gcg cct aat 289 Gly Leu Gln Ser Ser Asp Asp Phe Ala Leu Ile Val AsnAla Pro Asn 80 85 90 cat gaa gga ata cag gct gga gta gac aga ttc tat agaact tgc aaa 337 His Glu Gly Ile Gln Ala Gly Val Asp Arg Phe Tyr Arg ThrCys Lys 95 100 105 110 ctg gtc ggg atc aac atg agc aaa aag aag tcc tacata aat aga acc 385 Leu Val Gly Ile Asn Met Ser Lys Lys Lys Ser Tyr IleAsn Arg Thr 115 120 125 ggc aca ttc gaa ttc aca agc ttt ttc tac cgg tatggt ttt gtc gcc 433 Gly Thr Phe Glu Phe Thr Ser Phe Phe Tyr Arg Tyr GlyPhe Val Ala 130 135 140 aat ttc agc atg gag cta ccc agt ttt ggg gtt tccggg ata aat gaa 481 Asn Phe Ser Met Glu Leu Pro Ser Phe Gly Val Ser GlyIle Asn Glu 145 150 155 tct gca gac atg agc att gga atg aca gtt atc aaaaac aac atg ata 529 Ser Ala Asp Met Ser Ile Gly Met Thr Val Ile Lys AsnAsn Met Ile 160 165 170 aat aat gat ctc ggt ccc gcc acg gca caa atg gcactc caa ctc ttc 577 Asn Asn Asp Leu Gly Pro Ala Thr Ala Gln Met Ala LeuGln Leu Phe 175 180 185 190 att aag gat tat cgg tac aca tac cgg tgt caaaga ggc gat acc cag 625 Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg Cys Gln ArgGly Asp Thr Gln 195 200 205 ata caa acc aga aga tcc ttt gag ttg aag aaactg tgg gaa cag act 673 Ile Gln Thr Arg Arg Ser Phe Glu Leu Lys Lys LeuTrp Glu Gln Thr 210 215 220 cga tca aag act ggt cta ctg gta tca gat gggggt cca aac cta tac 721 Arg Ser Lys Thr Gly Leu Leu Val Ser Asp Gly GlyPro Asn Leu Tyr 225 230 235 aac atc aga aac cta cac atc ccg gaa gtc tgtttg aaa tgg gag ctg 769 Asn Ile Arg Asn Leu His Ile Pro Glu Val Cys LeuLys Trp Glu Leu 240 245 250 atg gat gaa gat tat aaa ggg agg cta tgt aatcca ttg aat cct ttc 817 Met Asp Glu Asp Tyr Lys Gly Arg Leu Cys Asn ProLeu Asn Pro Phe 255 260 265 270 gtt agc cac aaa gaa att gaa tca gtg aacagt gca gta gta atg cct 865 Val Ser His Lys Glu Ile Glu Ser Val Asn SerAla Val Val Met Pro 275 280 285 gcg cat ggc cct gcc aaa agc atg gag tatgat gct gtt gca aca aca 913 Ala His Gly Pro Ala Lys Ser Met Glu Tyr AspAla Val Ala Thr Thr 290 295 300 cac tct tgg atc ccc aag agg aac cgg tccata ttg aac aca agt caa 961 His Ser Trp Ile Pro Lys Arg Asn Arg Ser IleLeu Asn Thr Ser Gln 305 310 315 agg gga ata ctc gaa gat gag cag atg tatcag aaa tgc tgc aac ctg 1009 Arg Gly Ile Leu Glu Asp Glu Gln Met Tyr GlnLys Cys Cys Asn Leu 320 325 330 ttt gaa aaa ttc ttc ccc agc agc tca tacaga aga cca gtc gga att 1057 Phe Glu Lys Phe Phe Pro Ser Ser Ser Tyr ArgArg Pro Val Gly Ile 335 340 345 350 tct agt atg gtt gag gcc atg gtg tccagg gcc cgc att gat gca cga 1105 Ser Ser Met Val Glu Ala Met Val Ser ArgAla Arg Ile Asp Ala Arg 355 360 365 att gac ttc gaa tct gga cgg ata aagaag gat gag ttc gct gag atc 1153 Ile Asp Phe Glu Ser Gly Arg Ile Lys LysAsp Glu Phe Ala Glu Ile 370 375 380 atg aag atc tgt tcc acc att gaa gagctc aga cgg caa aaa 1195 Met Lys Ile Cys Ser Thr Ile Glu Glu Leu Arg ArgGln Lys 385 390 395 tagtgaattt agcttgatct tcgtgaaaaa atgccttgtt tctact1241 95 396 PRT Equine influenza virus H3N8 95 Met Lys Leu Arg Thr GlnIle Pro Ala Glu Met Leu Ala Ser Ile Asp 1 5 10 15 Leu Lys Tyr Phe AsnAsp Ser Thr Lys Lys Lys Ile Glu Lys Ile Arg 20 25 30 Pro Leu Leu Val AspGly Thr Ala Ser Leu Ser Pro Gly Met Met Met 35 40 45 Gly Met Phe Asn MetLeu Ser Thr Val Leu Gly Val Ser Ile Leu Asn 50 55 60 Leu Gly Gln Arg LysTyr Thr Lys Thr Thr Tyr Trp Trp Asp Gly Leu 65 70 75 80 Gln Ser Ser AspAsp Phe Ala Leu Ile Val Asn Ala Pro Asn His Glu 85 90 95 Gly Ile Gln AlaGly Val Asp Arg Phe Tyr Arg Thr Cys Lys Leu Val 100 105 110 Gly Ile AsnMet Ser Lys Lys Lys Ser Tyr Ile Asn Arg Thr Gly Thr 115 120 125 Phe GluPhe Thr Ser Phe Phe Tyr Arg Tyr Gly Phe Val Ala Asn Phe 130 135 140 SerMet Glu Leu Pro Ser Phe Gly Val Ser Gly Ile Asn Glu Ser Ala 145 150 155160 Asp Met Ser Ile Gly Met Thr Val Ile Lys Asn Asn Met Ile Asn Asn 165170 175 Asp Leu Gly Pro Ala Thr Ala Gln Met Ala Leu Gln Leu Phe Ile Lys180 185 190 Asp Tyr Arg Tyr Thr Tyr Arg Cys Gln Arg Gly Asp Thr Gln IleGln 195 200 205 Thr Arg Arg Ser Phe Glu Leu Lys Lys Leu Trp Glu Gln ThrArg Ser 210 215 220 Lys Thr Gly Leu Leu Val Ser Asp Gly Gly Pro Asn LeuTyr Asn Ile 225 230 235 240 Arg Asn Leu His Ile Pro Glu Val Cys Leu LysTrp Glu Leu Met Asp 245 250 255 Glu Asp Tyr Lys Gly Arg Leu Cys Asn ProLeu Asn Pro Phe Val Ser 260 265 270 His Lys Glu Ile Glu Ser Val Asn SerAla Val Val Met Pro Ala His 275 280 285 Gly Pro Ala Lys Ser Met Glu TyrAsp Ala Val Ala Thr Thr His Ser 290 295 300 Trp Ile Pro Lys Arg Asn ArgSer Ile Leu Asn Thr Ser Gln Arg Gly 305 310 315 320 Ile Leu Glu Asp GluGln Met Tyr Gln Lys Cys Cys Asn Leu Phe Glu 325 330 335 Lys Phe Phe ProSer Ser Ser Tyr Arg Arg Pro Val Gly Ile Ser Ser 340 345 350 Met Val GluAla Met Val Ser Arg Ala Arg Ile Asp Ala Arg Ile Asp 355 360 365 Phe GluSer Gly Arg Ile Lys Lys Asp Glu Phe Ala Glu Ile Met Lys 370 375 380 IleCys Ser Thr Ile Glu Glu Leu Arg Arg Gln Lys 385 390 395 96 1188 DNAEquine influenza virus H3N8 96 atgaaattga gaactcaaat accagcagaaatgctcgcaa gcattgatct gaaatatttc 60 aatgattcaa caaaaaagaa aattgagaagatacgaccac ttctggtcga tgggactgct 120 tcactgagtc ctggcatgat gatgggaatgttcaacatgt tgagcactgt actaggtgta 180 tccatattaa acctgggcca gaggaaatacacaaagacca catactggtg ggatggtctg 240 caatcatccg atgattttgc tttgatagtgaatgcgccta atcatgaagg aatacaggct 300 ggagtagaca gattctatag aacttgcaaactggtcggga tcaacatgag caaaaagaag 360 tcctacataa atagaaccgg cacattcgaattcacaagct ttttctaccg gtatggtttt 420 gtcgccaatt tcagcatgga gctacccagttttggggttt ccgggataaa tgaatctgca 480 gacatgagca ttggaatgac agttatcaaaaacaacatga taaataatga tctcggtccc 540 gccacggcac aaatggcact ccaactcttcattaaggatt atcggtacac ataccggtgt 600 caaagaggcg atacccagat acaaaccagaagatcctttg agttgaagaa actgtgggaa 660 cagactcgat caaagactgg tctactggtatcagatgggg gtccaaacct atacaacatc 720 agaaacctac acatcccgga agtctgtttgaaatgggagc tgatggatga agattataaa 780 gggaggctat gtaatccatt gaatcctttcgttagccaca aagaaattga atcagtgaac 840 agtgcagtag taatgcctgc gcatggccctgccaaaagca tggagtatga tgctgttgca 900 acaacacact cttggatccc caagaggaaccggtccatat tgaacacaag tcaaagggga 960 atactcgaag atgagcagat gtatcagaaatgctgcaacc tgtttgaaaa attcttcccc 1020 agcagctcat acagaagacc agtcggaatttctagtatgg ttgaggccat ggtgtccagg 1080 gcccgcattg atgcacgaat tgacttcgaatctggacgga taaagaagga tgagttcgct 1140 gagatcatga agatctgttc caccattgaagagctcagac ggcaaaaa 1188 97 20 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Primer 97 taaatagaac cggcacattc 20 98 17DNA Artificial Sequence Description of Artificial Sequence SyntheticPrimer 98 caaagaaatt gaatcag 17 99 18 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Primer 99 caagcattactactgcac 18 100 19 DNA Artificial Sequence Description of ArtificialSequence Synthetic Primer 100 agtctgttcc cacagtttc 19 101 20 DNAArtificial Sequence Description of Artificial Sequence Synthetic Primer101 gaattcgaat gtgccggttc 20 102 20 DNA Artificial Sequence Descriptionof Artificial Sequence Synthetic Primer 102 aaaacaagga ttttttcacg 20 1032341 DNA Equine influenza virus H3N8 CDS (25)..(2295) At 1489, w = a ort/unknown; At amino acid location 489, Xaa = unknown 103 agcaaaagcaggcaaactat ttga atg gat gtc aat ccg act cta ctc ttc 51 Met Asp Val AsnPro Thr Leu Leu Phe 1 5 tta aag gtg cca gcg caa aat gct ata agc aca acattc cct tat act 99 Leu Lys Val Pro Ala Gln Asn Ala Ile Ser Thr Thr PhePro Tyr Thr 10 15 20 25 gga gat cct ccc tac agt cat gga aca ggg aca ggatac acc atg gat 147 Gly Asp Pro Pro Tyr Ser His Gly Thr Gly Thr Gly TyrThr Met Asp 30 35 40 act gtc aac aga aca cat caa tac tca gaa aag ggg aaatgg aca aca 195 Thr Val Asn Arg Thr His Gln Tyr Ser Glu Lys Gly Lys TrpThr Thr 45 50 55 aac act gag att gga gca cca caa ctt aat cca atc gat ggaccg ctt 243 Asn Thr Glu Ile Gly Ala Pro Gln Leu Asn Pro Ile Asp Gly ProLeu 60 65 70 cct gaa gac aat gaa cca agt ggg tac gcc caa aca gat tgt gtattg 291 Pro Glu Asp Asn Glu Pro Ser Gly Tyr Ala Gln Thr Asp Cys Val Leu75 80 85 gaa gca atg gct ttc ctt gaa gaa tcc cat ccc gga atc ttt gaa aat339 Glu Ala Met Ala Phe Leu Glu Glu Ser His Pro Gly Ile Phe Glu Asn 9095 100 105 tcg tgt ctt gaa aca atg gag gtg gtt cag cag aca aga gtg gacaaa 387 Ser Cys Leu Glu Thr Met Glu Val Val Gln Gln Thr Arg Val Asp Lys110 115 120 cta aca caa ggc cga caa act tac gat tgg acc ttg aat agg aatcaa 435 Leu Thr Gln Gly Arg Gln Thr Tyr Asp Trp Thr Leu Asn Arg Asn Gln125 130 135 cct gcc gca aca gca ctt gct aat aca att gaa gtg ttc aga tcaaat 483 Pro Ala Ala Thr Ala Leu Ala Asn Thr Ile Glu Val Phe Arg Ser Asn140 145 150 gat ctg act tcc agt gag tca ggg aga tta atg gac ttc ctc aaagat 531 Asp Leu Thr Ser Ser Glu Ser Gly Arg Leu Met Asp Phe Leu Lys Asp155 160 165 gtc atg gag tcc atg aac aag gaa gaa atg gaa ata aca aca cacttc 579 Val Met Glu Ser Met Asn Lys Glu Glu Met Glu Ile Thr Thr His Phe170 175 180 185 caa cgg aag aga aga gta aga gac aac atg aca aag aga atggtg aca 627 Gln Arg Lys Arg Arg Val Arg Asp Asn Met Thr Lys Arg Met ValThr 190 195 200 cag aga acc ata ggg aag aaa aaa caa cga tta aac aga aagagc tat 675 Gln Arg Thr Ile Gly Lys Lys Lys Gln Arg Leu Asn Arg Lys SerTyr 205 210 215 ctg atc agg gca tta acc tta aac aca atg acc aag gac gctgag aga 723 Leu Ile Arg Ala Leu Thr Leu Asn Thr Met Thr Lys Asp Ala GluArg 220 225 230 ggg aaa ttg aaa cga cga gca att gca acc cca gga atg cagata aga 771 Gly Lys Leu Lys Arg Arg Ala Ile Ala Thr Pro Gly Met Gln IleArg 235 240 245 ggg ttt gta tat ttt gtt gaa aca tta gcc cga aga ata tgtgaa aag 819 Gly Phe Val Tyr Phe Val Glu Thr Leu Ala Arg Arg Ile Cys GluLys 250 255 260 265 ctt gaa caa tca gga ttg cca gtt ggc ggt aat gag aaaaag gcc aaa 867 Leu Glu Gln Ser Gly Leu Pro Val Gly Gly Asn Glu Lys LysAla Lys 270 275 280 ctg gct aat gtc gtc aga aaa atg atg act aat tcc caagac act gaa 915 Leu Ala Asn Val Val Arg Lys Met Met Thr Asn Ser Gln AspThr Glu 285 290 295 ctc tcc ttc acc atc act ggg gac aat acc aaa tgg aatgaa aat cag 963 Leu Ser Phe Thr Ile Thr Gly Asp Asn Thr Lys Trp Asn GluAsn Gln 300 305 310 aac cca cgc atg ttc ctg gca atg atc aca tac ata actaga aac cag 1011 Asn Pro Arg Met Phe Leu Ala Met Ile Thr Tyr Ile Thr ArgAsn Gln 315 320 325 cca gaa tgg ttc aga aat gtt cta agc att gca ccg attatg ttc tca 1059 Pro Glu Trp Phe Arg Asn Val Leu Ser Ile Ala Pro Ile MetPhe Ser 330 335 340 345 aat aaa atg gca aga ctg ggg aaa gga tat atg tttgaa agc aaa agt 1107 Asn Lys Met Ala Arg Leu Gly Lys Gly Tyr Met Phe GluSer Lys Ser 350 355 360 atg aaa ttg aga act caa ata cca gca gaa atg ctcgca agc att gat 1155 Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met Leu AlaSer Ile Asp 365 370 375 ctg aaa tat ttc aat gat tca aca aaa aag aaa attgag aag ata cga 1203 Leu Lys Tyr Phe Asn Asp Ser Thr Lys Lys Lys Ile GluLys Ile Arg 380 385 390 cca ctt ctg gtc gat ggg act gct tca ctg agt cctggc atg atg atg 1251 Pro Leu Leu Val Asp Gly Thr Ala Ser Leu Ser Pro GlyMet Met Met 395 400 405 gga atg ttc aac atg ttg agc act gta cta ggt gtatcc ata tta aac 1299 Gly Met Phe Asn Met Leu Ser Thr Val Leu Gly Val SerIle Leu Asn 410 415 420 425 ctg ggc cag agg aaa tac aca aag acc aca tactgg tgg gat ggt ctg 1347 Leu Gly Gln Arg Lys Tyr Thr Lys Thr Thr Tyr TrpTrp Asp Gly Leu 430 435 440 caa tca tcc gat gat ttt gct ttg ata gtg aatgcg cct aat cat gaa 1395 Gln Ser Ser Asp Asp Phe Ala Leu Ile Val Asn AlaPro Asn His Glu 445 450 455 gga ata cag gct gga gta gac aga ttc tat agaact tgc aaa ctg gtc 1443 Gly Ile Gln Ala Gly Val Asp Arg Phe Tyr Arg ThrCys Lys Leu Val 460 465 470 ggg atc aac atg agc aaa aag aag tcc tac ataaat aga acc ggc wca 1491 Gly Ile Asn Met Ser Lys Lys Lys Ser Tyr Ile AsnArg Thr Gly Xaa 475 480 485 ttc gaa ttc aca agc ttt ttc tac cgg tat ggtttt gtc gcc aat ttc 1539 Phe Glu Phe Thr Ser Phe Phe Tyr Arg Tyr Gly PheVal Ala Asn Phe 490 495 500 505 agc atg gag cta ccc agt ttt ggg gtt tccggg ata aat gaa tct gca 1587 Ser Met Glu Leu Pro Ser Phe Gly Val Ser GlyIle Asn Glu Ser Ala 510 515 520 gac atg agc att gga atg aca gtt atc aaaaac aac atg ata aat aat 1635 Asp Met Ser Ile Gly Met Thr Val Ile Lys AsnAsn Met Ile Asn Asn 525 530 535 gat ctc ggt ccc gcc acg gca caa atg gcactc caa ctc ttc att aag 1683 Asp Leu Gly Pro Ala Thr Ala Gln Met Ala LeuGln Leu Phe Ile Lys 540 545 550 gat tat cgg tac aca tac cgg tgc cat agaggc gat acc cag ata caa 1731 Asp Tyr Arg Tyr Thr Tyr Arg Cys His Arg GlyAsp Thr Gln Ile Gln 555 560 565 acc aga aga tcc ttt gag ttg aag aaa ctgtgg gaa cag act cga tca 1779 Thr Arg Arg Ser Phe Glu Leu Lys Lys Leu TrpGlu Gln Thr Arg Ser 570 575 580 585 aag act ggt cta ctg gta tca gat gggggt cca aac cta tac aac atc 1827 Lys Thr Gly Leu Leu Val Ser Asp Gly GlyPro Asn Leu Tyr Asn Ile 590 595 600 aga aac cta cac atc ccg gaa gtc tgtttg aaa tgg gag ctg atg gat 1875 Arg Asn Leu His Ile Pro Glu Val Cys LeuLys Trp Glu Leu Met Asp 605 610 615 gaa gat tat aaa ggg agg cta tgt aatcca ttg aat cct ttc gtt agc 1923 Glu Asp Tyr Lys Gly Arg Leu Cys Asn ProLeu Asn Pro Phe Val Ser 620 625 630 cac aaa gaa att gaa tca gtg aac agtgca gta gta atg cct gcg cat 1971 His Lys Glu Ile Glu Ser Val Asn Ser AlaVal Val Met Pro Ala His 635 640 645 ggc cct gcc aaa agc atg gag tat gatgct gtt gca aca aca cac tct 2019 Gly Pro Ala Lys Ser Met Glu Tyr Asp AlaVal Ala Thr Thr His Ser 650 655 660 665 tgg atc ccc aag agg aac cgg tccata ttg aac aca agt caa agg gga 2067 Trp Ile Pro Lys Arg Asn Arg Ser IleLeu Asn Thr Ser Gln Arg Gly 670 675 680 ata ctc gaa gat gag cag atg tatcag aaa tgc tgc aac ctg ttt gaa 2115 Ile Leu Glu Asp Glu Gln Met Tyr GlnLys Cys Cys Asn Leu Phe Glu 685 690 695 aaa ttc ttc ccc agc agc tca tacaga aga cca gtc gga att tct agt 2163 Lys Phe Phe Pro Ser Ser Ser Tyr ArgArg Pro Val Gly Ile Ser Ser 700 705 710 atg gtt gag gcc atg gtg tcc agggcc cgc att gat gca cga att gac 2211 Met Val Glu Ala Met Val Ser Arg AlaArg Ile Asp Ala Arg Ile Asp 715 720 725 ttc gaa tct gga cgg ata aag aaggat gag ttc gct gag atc atg aag 2259 Phe Glu Ser Gly Arg Ile Lys Lys AspGlu Phe Ala Glu Ile Met Lys 730 735 740 745 atc tgt tcc acc att gaa gagctc aga cgg caa aaa tagtgaattt 2305 Ile Cys Ser Thr Ile Glu Glu Leu ArgArg Gln Lys 750 755 agcttgatct tcgtgaaaaa atgccttgtt tctact 2341 104 757PRT Equine influenza virus H3N8 At amino acid location 489, Xaa =unknown 104 Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala GlnAsn 1 5 10 15 Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro TyrSer His 20 25 30 Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg ThrHis Gln 35 40 45 Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu Ile GlyAla Pro 50 55 60 Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp Asn GluPro Ser 65 70 75 80 Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu Ala Met AlaPhe Leu Glu 85 90 95 Glu Ser His Pro Gly Ile Phe Glu Asn Ser Cys Leu GluThr Met Glu 100 105 110 Val Val Gln Gln Thr Arg Val Asp Lys Leu Thr GlnGly Arg Gln Thr 115 120 125 Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro AlaAla Thr Ala Leu Ala 130 135 140 Asn Thr Ile Glu Val Phe Arg Ser Asn AspLeu Thr Ser Ser Glu Ser 145 150 155 160 Gly Arg Leu Met Asp Phe Leu LysAsp Val Met Glu Ser Met Asn Lys 165 170 175 Glu Glu Met Glu Ile Thr ThrHis Phe Gln Arg Lys Arg Arg Val Arg 180 185 190 Asp Asn Met Thr Lys ArgMet Val Thr Gln Arg Thr Ile Gly Lys Lys 195 200 205 Lys Gln Arg Leu AsnArg Lys Ser Tyr Leu Ile Arg Ala Leu Thr Leu 210 215 220 Asn Thr Met ThrLys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala 225 230 235 240 Ile AlaThr Pro Gly Met Gln Ile Arg Gly Phe Val Tyr Phe Val Glu 245 250 255 ThrLeu Ala Arg Arg Ile Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro 260 265 270Val Gly Gly Asn Glu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys 275 280285 Met Met Thr Asn Ser Gln Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly 290295 300 Asp Asn Thr Lys Trp Asn Glu Asn Gln Asn Pro Arg Met Phe Leu Ala305 310 315 320 Met Ile Thr Tyr Ile Thr Arg Asn Gln Pro Glu Trp Phe ArgAsn Val 325 330 335 Leu Ser Ile Ala Pro Ile Met Phe Ser Asn Lys Met AlaArg Leu Gly 340 345 350 Lys Gly Tyr Met Phe Glu Ser Lys Ser Met Lys LeuArg Thr Gln Ile 355 360 365 Pro Ala Glu Met Leu Ala Ser Ile Asp Leu LysTyr Phe Asn Asp Ser 370 375 380 Thr Lys Lys Lys Ile Glu Lys Ile Arg ProLeu Leu Val Asp Gly Thr 385 390 395 400 Ala Ser Leu Ser Pro Gly Met MetMet Gly Met Phe Asn Met Leu Ser 405 410 415 Thr Val Leu Gly Val Ser IleLeu Asn Leu Gly Gln Arg Lys Tyr Thr 420 425 430 Lys Thr Thr Tyr Trp TrpAsp Gly Leu Gln Ser Ser Asp Asp Phe Ala 435 440 445 Leu Ile Val Asn AlaPro Asn His Glu Gly Ile Gln Ala Gly Val Asp 450 455 460 Arg Phe Tyr ArgThr Cys Lys Leu Val Gly Ile Asn Met Ser Lys Lys 465 470 475 480 Lys SerTyr Ile Asn Arg Thr Gly Xaa Phe Glu Phe Thr Ser Phe Phe 485 490 495 TyrArg Tyr Gly Phe Val Ala Asn Phe Ser Met Glu Leu Pro Ser Phe 500 505 510Gly Val Ser Gly Ile Asn Glu Ser Ala Asp Met Ser Ile Gly Met Thr 515 520525 Val Ile Lys Asn Asn Met Ile Asn Asn Asp Leu Gly Pro Ala Thr Ala 530535 540 Gln Met Ala Leu Gln Leu Phe Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg545 550 555 560 Cys His Arg Gly Asp Thr Gln Ile Gln Thr Arg Arg Ser PheGlu Leu 565 570 575 Lys Lys Leu Trp Glu Gln Thr Arg Ser Lys Thr Gly LeuLeu Val Ser 580 585 590 Asp Gly Gly Pro Asn Leu Tyr Asn Ile Arg Asn LeuHis Ile Pro Glu 595 600 605 Val Cys Leu Lys Trp Glu Leu Met Asp Glu AspTyr Lys Gly Arg Leu 610 615 620 Cys Asn Pro Leu Asn Pro Phe Val Ser HisLys Glu Ile Glu Ser Val 625 630 635 640 Asn Ser Ala Val Val Met Pro AlaHis Gly Pro Ala Lys Ser Met Glu 645 650 655 Tyr Asp Ala Val Ala Thr ThrHis Ser Trp Ile Pro Lys Arg Asn Arg 660 665 670 Ser Ile Leu Asn Thr SerGln Arg Gly Ile Leu Glu Asp Glu Gln Met 675 680 685 Tyr Gln Lys Cys CysAsn Leu Phe Glu Lys Phe Phe Pro Ser Ser Ser 690 695 700 Tyr Arg Arg ProVal Gly Ile Ser Ser Met Val Glu Ala Met Val Ser 705 710 715 720 Arg AlaArg Ile Asp Ala Arg Ile Asp Phe Glu Ser Gly Arg Ile Lys 725 730 735 LysAsp Glu Phe Ala Glu Ile Met Lys Ile Cys Ser Thr Ile Glu Glu 740 745 750Leu Arg Arg Gln Lys 755 105 2271 DNA Equine influenza virus H3N8 105atggatgtca atccgactct actcttctta aaggtgccag cgcaaaatgc tataagcaca 60acattccctt atactggaga tcctccctac agtcatggaa cagggacagg atacaccatg 120gatactgtca acagaacaca tcaatactca gaaaagggga aatggacaac aaacactgag 180attggagcac cacaacttaa tccaatcgat ggaccgcttc ctgaagacaa tgaaccaagt 240gggtacgccc aaacagattg tgtattggaa gcaatggctt tccttgaaga atcccatccc 300ggaatctttg aaaattcgtg tcttgaaaca atggaggtgg ttcagcagac aagagtggac 360aaactaacac aaggccgaca aacttacgat tggaccttga ataggaatca acctgccgca 420acagcacttg ctaatacaat tgaagtgttc agatcaaatg atctgacttc cagtgagtca 480gggagattaa tggacttcct caaagatgtc atggagtcca tgaacaagga agaaatggaa 540ataacaacac acttccaacg gaagagaaga gtaagagaca acatgacaaa gagaatggtg 600acacagagaa ccatagggaa gaaaaaacaa cgattaaaca gaaagagcta tctgatcagg 660gcattaacct taaacacaat gaccaaggac gctgagagag ggaaattgaa acgacgagca 720attgcaaccc caggaatgca gataagaggg tttgtatatt ttgttgaaac attagcccga 780agaatatgtg aaaagcttga acaatcagga ttgccagttg gcggtaatga gaaaaaggcc 840aaactggcta atgtcgtcag aaaaatgatg actaattccc aagacactga actctccttc 900accatcactg gggacaatac caaatggaat gaaaatcaga acccacgcat gttcctggca 960atgatcacat acataactag aaaccagcca gaatggttca gaaatgttct aagcattgca 1020ccgattatgt tctcaaataa aatggcaaga ctggggaaag gatatatgtt tgaaagcaaa 1080agtatgaaat tgagaactca aataccagca gaaatgctcg caagcattga tctgaaatat 1140ttcaatgatt caacaaaaaa gaaaattgag aagatacgac cacttctggt cgatgggact 1200gcttcactga gtcctggcat gatgatggga atgttcaaca tgttgagcac tgtactaggt 1260gtatccatat taaacctggg ccagaggaaa tacacaaaga ccacatactg gtgggatggt 1320ctgcaatcat ccgatgattt tgctttgata gtgaatgcgc ctaatcatga aggaatacag 1380gctggagtag acagattcta tagaacttgc aaactggtcg ggatcaacat gagcaaaaag 1440aagtcctaca taaatagaac cggcwcattc gaattcacaa gctttttcta ccggtatggt 1500tttgtcgcca atttcagcat ggagctaccc agttttgggg tttccgggat aaatgaatct 1560gcagacatga gcattggaat gacagttatc aaaaacaaca tgataaataa tgatctcggt 1620cccgccacgg cacaaatggc actccaactc ttcattaagg attatcggta cacataccgg 1680tgccatagag gcgataccca gatacaaacc agaagatcct ttgagttgaa gaaactgtgg 1740gaacagactc gatcaaagac tggtctactg gtatcagatg ggggtccaaa cctatacaac 1800atcagaaacc tacacatccc ggaagtctgt ttgaaatggg agctgatgga tgaagattat 1860aaagggaggc tatgtaatcc attgaatcct ttcgttagcc acaaagaaat tgaatcagtg 1920aacagtgcag tagtaatgcc tgcgcatggc cctgccaaaa gcatggagta tgatgctgtt 1980gcaacaacac actcttggat ccccaagagg aaccggtcca tattgaacac aagtcaaagg 2040ggaatactcg aagatgagca gatgtatcag aaatgctgca acctgtttga aaaattcttc 2100cccagcagct catacagaag accagtcgga atttctagta tggttgaggc catggtgtcc 2160agggcccgca ttgatgcacg aattgacttc gaatctggac ggataaagaa ggatgagttc 2220gctgagatca tgaagatctg ttccaccatt gaagagctca gacggcaaaa a 2271 106 2341DNA Equine influenza virus H3N8 CDS (25)..(2295) At 2144, r = g or a. Atamino acid location 707, Xaa = unknown 106 agcaaaagca ggcaaactat ttgaatg gat gtc aat ccg act cta ctc ttc 51 Met Asp Val Asn Pro Thr Leu LeuPhe 1 5 tta aag gtg cca gcg caa aat gct ata agc aca aca ttc cct tat act99 Leu Lys Val Pro Ala Gln Asn Ala Ile Ser Thr Thr Phe Pro Tyr Thr 10 1520 25 gga gat cct ccc tac agt cat gga aca ggg aca gga tac acc atg gat147 Gly Asp Pro Pro Tyr Ser His Gly Thr Gly Thr Gly Tyr Thr Met Asp 3035 40 act gtc aac aga aca cat caa tac tca gaa aag ggg aaa tgg aca aca195 Thr Val Asn Arg Thr His Gln Tyr Ser Glu Lys Gly Lys Trp Thr Thr 4550 55 aac act gag att gga gca cca caa ctt aat cca atc gat gga ccg ctt243 Asn Thr Glu Ile Gly Ala Pro Gln Leu Asn Pro Ile Asp Gly Pro Leu 6065 70 cct gaa gac aat gaa cca agt ggg tac gcc caa aca gat tgt gta ttg291 Pro Glu Asp Asn Glu Pro Ser Gly Tyr Ala Gln Thr Asp Cys Val Leu 7580 85 gaa gca atg gct ttc ctt gaa gaa tcc cat ccc gga atc ttt gaa aat339 Glu Ala Met Ala Phe Leu Glu Glu Ser His Pro Gly Ile Phe Glu Asn 9095 100 105 tcg tgt ctt gaa aca atg gag gtg gtt cag cag aca aga gtg gacaaa 387 Ser Cys Leu Glu Thr Met Glu Val Val Gln Gln Thr Arg Val Asp Lys110 115 120 cta aca caa ggc cga caa act tac gat tgg acc ttg aat agg aatcaa 435 Leu Thr Gln Gly Arg Gln Thr Tyr Asp Trp Thr Leu Asn Arg Asn Gln125 130 135 cct gcc gca aca gca ctt gct aat aca att gaa gtg ttc aga tcaaat 483 Pro Ala Ala Thr Ala Leu Ala Asn Thr Ile Glu Val Phe Arg Ser Asn140 145 150 gat ctg act tcc agt gag tca ggg aga tta atg gac ttc ctc aaagat 531 Asp Leu Thr Ser Ser Glu Ser Gly Arg Leu Met Asp Phe Leu Lys Asp155 160 165 gtc atg gag tcc atg aac aag gaa gaa atg gaa ata aca aca cacttc 579 Val Met Glu Ser Met Asn Lys Glu Glu Met Glu Ile Thr Thr His Phe170 175 180 185 caa cgg aag aga aga gta aga gac aac atg aca aag aga atggtg aca 627 Gln Arg Lys Arg Arg Val Arg Asp Asn Met Thr Lys Arg Met ValThr 190 195 200 cag aga acc ata ggg aag aaa aaa caa cga tta aac aga aagagc tat 675 Gln Arg Thr Ile Gly Lys Lys Lys Gln Arg Leu Asn Arg Lys SerTyr 205 210 215 ctg atc agg gca tta acc tta aac aca atg acc aag gac gctgag aga 723 Leu Ile Arg Ala Leu Thr Leu Asn Thr Met Thr Lys Asp Ala GluArg 220 225 230 ggg aaa ttg aaa cga cga gca att gca acc cca gga atg cagata aga 771 Gly Lys Leu Lys Arg Arg Ala Ile Ala Thr Pro Gly Met Gln IleArg 235 240 245 ggg ttt gta tat ttt gtt gaa aca tta gcc cga aga ata tgtgaa aag 819 Gly Phe Val Tyr Phe Val Glu Thr Leu Ala Arg Arg Ile Cys GluLys 250 255 260 265 ctt gaa caa tca gga ttg cca gtt ggc ggt aat gag aaaaag gcc aaa 867 Leu Glu Gln Ser Gly Leu Pro Val Gly Gly Asn Glu Lys LysAla Lys 270 275 280 ctg gct aat gtc gtc aga aaa atg atg act aat tcc caagac act gaa 915 Leu Ala Asn Val Val Arg Lys Met Met Thr Asn Ser Gln AspThr Glu 285 290 295 ctc tcc ttc acc atc act ggg gac aat acc aaa tgg aatgaa aat cag 963 Leu Ser Phe Thr Ile Thr Gly Asp Asn Thr Lys Trp Asn GluAsn Gln 300 305 310 aac cca cgc atg ttc ctg gca atg atc aca tac ata actaga aac cag 1011 Asn Pro Arg Met Phe Leu Ala Met Ile Thr Tyr Ile Thr ArgAsn Gln 315 320 325 cca gaa tgg ttc aga aat gtt cta agc att gca ccg attatg ttc tca 1059 Pro Glu Trp Phe Arg Asn Val Leu Ser Ile Ala Pro Ile MetPhe Ser 330 335 340 345 aat aaa atg gca aga ctg ggg aaa gga tat atg tttgaa agc aaa agt 1107 Asn Lys Met Ala Arg Leu Gly Lys Gly Tyr Met Phe GluSer Lys Ser 350 355 360 atg aaa ttg aga act caa ata cca gca gaa atg ctcgca agc att gat 1155 Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met Leu AlaSer Ile Asp 365 370 375 ctg aaa tat ttc aat gat tca aca aaa aag aaa attgag aag ata cga 1203 Leu Lys Tyr Phe Asn Asp Ser Thr Lys Lys Lys Ile GluLys Ile Arg 380 385 390 cca ctt ctg gtc gat ggg act gct tca ctg agt cctggc atg atg atg 1251 Pro Leu Leu Val Asp Gly Thr Ala Ser Leu Ser Pro GlyMet Met Met 395 400 405 gga atg ttc aac atg ttg agc act gta cta ggt gtatcc ata tta aac 1299 Gly Met Phe Asn Met Leu Ser Thr Val Leu Gly Val SerIle Leu Asn 410 415 420 425 ctg ggc cag agg aaa tac aca aag acc aca tactgg tgg gat ggt ctg 1347 Leu Gly Gln Arg Lys Tyr Thr Lys Thr Thr Tyr TrpTrp Asp Gly Leu 430 435 440 caa tca tcc gat gat ttt gct ttg ata gtg aatgcg cct aat cat gaa 1395 Gln Ser Ser Asp Asp Phe Ala Leu Ile Val Asn AlaPro Asn His Glu 445 450 455 gga ata cag gct gga gta gac aga ttc tat agaact tgc aaa ctg gtc 1443 Gly Ile Gln Ala Gly Val Asp Arg Phe Tyr Arg ThrCys Lys Leu Val 460 465 470 ggg atc aac atg agc aaa aag aag tcc tac ataaat aga acc ggc aca 1491 Gly Ile Asn Met Ser Lys Lys Lys Ser Tyr Ile AsnArg Thr Gly Thr 475 480 485 ttc gaa ttc aca agc ttt ttc tac cgg tat ggtttt gtc gcc aat ttc 1539 Phe Glu Phe Thr Ser Phe Phe Tyr Arg Tyr Gly PheVal Ala Asn Phe 490 495 500 505 agc atg gag cta ccc agt ttt ggg gtt tccggg ata aat gaa tct gca 1587 Ser Met Glu Leu Pro Ser Phe Gly Val Ser GlyIle Asn Glu Ser Ala 510 515 520 gac atg agc att gga atg aca gtt atc aaaaac aac atg ata aat aat 1635 Asp Met Ser Ile Gly Met Thr Val Ile Lys AsnAsn Met Ile Asn Asn 525 530 535 gat ctc ggt ccc gcc acg gca caa atg gcactc caa ctc ttc att aag 1683 Asp Leu Gly Pro Ala Thr Ala Gln Met Ala LeuGln Leu Phe Ile Lys 540 545 550 gat tat cgg tac aca tac cgg tgt caa agaggc gat acc cag ata caa 1731 Asp Tyr Arg Tyr Thr Tyr Arg Cys Gln Arg GlyAsp Thr Gln Ile Gln 555 560 565 acc aga aga tcc ttt gag ttg aag aaa ctgtgg gaa cag act cga tca 1779 Thr Arg Arg Ser Phe Glu Leu Lys Lys Leu TrpGlu Gln Thr Arg Ser 570 575 580 585 aag act ggt cta ctg gta tca gat gggggt cca aac cta tac aac atc 1827 Lys Thr Gly Leu Leu Val Ser Asp Gly GlyPro Asn Leu Tyr Asn Ile 590 595 600 aga aac cta cac atc ccg gaa gtc tgtttg aaa tgg gag ctg atg gat 1875 Arg Asn Leu His Ile Pro Glu Val Cys LeuLys Trp Glu Leu Met Asp 605 610 615 gaa gat tat aaa ggg agg cta tgt aatcca ttg aat cct ttc gtt agc 1923 Glu Asp Tyr Lys Gly Arg Leu Cys Asn ProLeu Asn Pro Phe Val Ser 620 625 630 cac aaa gaa att gaa tca gtg aac agtgca gta gta atg cct gcg cat 1971 His Lys Glu Ile Glu Ser Val Asn Ser AlaVal Val Met Pro Ala His 635 640 645 ggc cct gcc aaa agc atg gag tat gatgct gtt gca aca aca cac tct 2019 Gly Pro Ala Lys Ser Met Glu Tyr Asp AlaVal Ala Thr Thr His Ser 650 655 660 665 tgg atc ccc aag agg aac cgg tccata ttg aac aca agt caa agg gga 2067 Trp Ile Pro Lys Arg Asn Arg Ser IleLeu Asn Thr Ser Gln Arg Gly 670 675 680 ata ctc gaa gat gag cag atg tatcag aaa tgc tgc aac ctg ttt gaa 2115 Ile Leu Glu Asp Glu Gln Met Tyr GlnLys Cys Cys Asn Leu Phe Glu 685 690 695 aaa ttc ttc ccc agc agc tca tacaga ara cca gtc gga att tct agt 2163 Lys Phe Phe Pro Ser Ser Ser Tyr ArgXaa Pro Val Gly Ile Ser Ser 700 705 710 atg gtt gag gcc atg gtg tcc agggcc cgc att gat gca cga att gac 2211 Met Val Glu Ala Met Val Ser Arg AlaArg Ile Asp Ala Arg Ile Asp 715 720 725 ttc gaa tct gga cgg ata aag aaggat gag ttc gct gag atc atg aag 2259 Phe Glu Ser Gly Arg Ile Lys Lys AspGlu Phe Ala Glu Ile Met Lys 730 735 740 745 atc tgt tcc acc att gaa gagctc aga cgg caa aaa tagtgaattt 2305 Ile Cys Ser Thr Ile Glu Glu Leu ArgArg Gln Lys 750 755 agcttgatct tcgtgaaaaa atgccttgtt tctact 2341 107 757PRT Equine influenza virus H3N8 At amino acid location 707, Xaa =unknown 107 Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala GlnAsn 1 5 10 15 Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro TyrSer His 20 25 30 Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg ThrHis Gln 35 40 45 Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu Ile GlyAla Pro 50 55 60 Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp Asn GluPro Ser 65 70 75 80 Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu Ala Met AlaPhe Leu Glu 85 90 95 Glu Ser His Pro Gly Ile Phe Glu Asn Ser Cys Leu GluThr Met Glu 100 105 110 Val Val Gln Gln Thr Arg Val Asp Lys Leu Thr GlnGly Arg Gln Thr 115 120 125 Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro AlaAla Thr Ala Leu Ala 130 135 140 Asn Thr Ile Glu Val Phe Arg Ser Asn AspLeu Thr Ser Ser Glu Ser 145 150 155 160 Gly Arg Leu Met Asp Phe Leu LysAsp Val Met Glu Ser Met Asn Lys 165 170 175 Glu Glu Met Glu Ile Thr ThrHis Phe Gln Arg Lys Arg Arg Val Arg 180 185 190 Asp Asn Met Thr Lys ArgMet Val Thr Gln Arg Thr Ile Gly Lys Lys 195 200 205 Lys Gln Arg Leu AsnArg Lys Ser Tyr Leu Ile Arg Ala Leu Thr Leu 210 215 220 Asn Thr Met ThrLys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala 225 230 235 240 Ile AlaThr Pro Gly Met Gln Ile Arg Gly Phe Val Tyr Phe Val Glu 245 250 255 ThrLeu Ala Arg Arg Ile Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro 260 265 270Val Gly Gly Asn Glu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys 275 280285 Met Met Thr Asn Ser Gln Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly 290295 300 Asp Asn Thr Lys Trp Asn Glu Asn Gln Asn Pro Arg Met Phe Leu Ala305 310 315 320 Met Ile Thr Tyr Ile Thr Arg Asn Gln Pro Glu Trp Phe ArgAsn Val 325 330 335 Leu Ser Ile Ala Pro Ile Met Phe Ser Asn Lys Met AlaArg Leu Gly 340 345 350 Lys Gly Tyr Met Phe Glu Ser Lys Ser Met Lys LeuArg Thr Gln Ile 355 360 365 Pro Ala Glu Met Leu Ala Ser Ile Asp Leu LysTyr Phe Asn Asp Ser 370 375 380 Thr Lys Lys Lys Ile Glu Lys Ile Arg ProLeu Leu Val Asp Gly Thr 385 390 395 400 Ala Ser Leu Ser Pro Gly Met MetMet Gly Met Phe Asn Met Leu Ser 405 410 415 Thr Val Leu Gly Val Ser IleLeu Asn Leu Gly Gln Arg Lys Tyr Thr 420 425 430 Lys Thr Thr Tyr Trp TrpAsp Gly Leu Gln Ser Ser Asp Asp Phe Ala 435 440 445 Leu Ile Val Asn AlaPro Asn His Glu Gly Ile Gln Ala Gly Val Asp 450 455 460 Arg Phe Tyr ArgThr Cys Lys Leu Val Gly Ile Asn Met Ser Lys Lys 465 470 475 480 Lys SerTyr Ile Asn Arg Thr Gly Thr Phe Glu Phe Thr Ser Phe Phe 485 490 495 TyrArg Tyr Gly Phe Val Ala Asn Phe Ser Met Glu Leu Pro Ser Phe 500 505 510Gly Val Ser Gly Ile Asn Glu Ser Ala Asp Met Ser Ile Gly Met Thr 515 520525 Val Ile Lys Asn Asn Met Ile Asn Asn Asp Leu Gly Pro Ala Thr Ala 530535 540 Gln Met Ala Leu Gln Leu Phe Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg545 550 555 560 Cys Gln Arg Gly Asp Thr Gln Ile Gln Thr Arg Arg Ser PheGlu Leu 565 570 575 Lys Lys Leu Trp Glu Gln Thr Arg Ser Lys Thr Gly LeuLeu Val Ser 580 585 590 Asp Gly Gly Pro Asn Leu Tyr Asn Ile Arg Asn LeuHis Ile Pro Glu 595 600 605 Val Cys Leu Lys Trp Glu Leu Met Asp Glu AspTyr Lys Gly Arg Leu 610 615 620 Cys Asn Pro Leu Asn Pro Phe Val Ser HisLys Glu Ile Glu Ser Val 625 630 635 640 Asn Ser Ala Val Val Met Pro AlaHis Gly Pro Ala Lys Ser Met Glu 645 650 655 Tyr Asp Ala Val Ala Thr ThrHis Ser Trp Ile Pro Lys Arg Asn Arg 660 665 670 Ser Ile Leu Asn Thr SerGln Arg Gly Ile Leu Glu Asp Glu Gln Met 675 680 685 Tyr Gln Lys Cys CysAsn Leu Phe Glu Lys Phe Phe Pro Ser Ser Ser 690 695 700 Tyr Arg Xaa ProVal Gly Ile Ser Ser Met Val Glu Ala Met Val Ser 705 710 715 720 Arg AlaArg Ile Asp Ala Arg Ile Asp Phe Glu Ser Gly Arg Ile Lys 725 730 735 LysAsp Glu Phe Ala Glu Ile Met Lys Ile Cys Ser Thr Ile Glu Glu 740 745 750Leu Arg Arg Gln Lys 755 108 2271 DNA Equine influenza virus H3N8 108atggatgtca atccgactct actcttctta aaggtgccag cgcaaaatgc tataagcaca 60acattccctt atactggaga tcctccctac agtcatggaa cagggacagg atacaccatg 120gatactgtca acagaacaca tcaatactca gaaaagggga aatggacaac aaacactgag 180attggagcac cacaacttaa tccaatcgat ggaccgcttc ctgaagacaa tgaaccaagt 240gggtacgccc aaacagattg tgtattggaa gcaatggctt tccttgaaga atcccatccc 300ggaatctttg aaaattcgtg tcttgaaaca atggaggtgg ttcagcagac aagagtggac 360aaactaacac aaggccgaca aacttacgat tggaccttga ataggaatca acctgccgca 420acagcacttg ctaatacaat tgaagtgttc agatcaaatg atctgacttc cagtgagtca 480gggagattaa tggacttcct caaagatgtc atggagtcca tgaacaagga agaaatggaa 540ataacaacac acttccaacg gaagagaaga gtaagagaca acatgacaaa gagaatggtg 600acacagagaa ccatagggaa gaaaaaacaa cgattaaaca gaaagagcta tctgatcagg 660gcattaacct taaacacaat gaccaaggac gctgagagag ggaaattgaa acgacgagca 720attgcaaccc caggaatgca gataagaggg tttgtatatt ttgttgaaac attagcccga 780agaatatgtg aaaagcttga acaatcagga ttgccagttg gcggtaatga gaaaaaggcc 840aaactggcta atgtcgtcag aaaaatgatg actaattccc aagacactga actctccttc 900accatcactg gggacaatac caaatggaat gaaaatcaga acccacgcat gttcctggca 960atgatcacat acataactag aaaccagcca gaatggttca gaaatgttct aagcattgca 1020ccgattatgt tctcaaataa aatggcaaga ctggggaaag gatatatgtt tgaaagcaaa 1080agtatgaaat tgagaactca aataccagca gaaatgctcg caagcattga tctgaaatat 1140ttcaatgatt caacaaaaaa gaaaattgag aagatacgac cacttctggt cgatgggact 1200gcttcactga gtcctggcat gatgatggga atgttcaaca tgttgagcac tgtactaggt 1260gtatccatat taaacctggg ccagaggaaa tacacaaaga ccacatactg gtgggatggt 1320ctgcaatcat ccgatgattt tgctttgata gtgaatgcgc ctaatcatga aggaatacag 1380gctggagtag acagattcta tagaacttgc aaactggtcg ggatcaacat gagcaaaaag 1440aagtcctaca taaatagaac cggcacattc gaattcacaa gctttttcta ccggtatggt 1500tttgtcgcca atttcagcat ggagctaccc agttttgggg tttccgggat aaatgaatct 1560gcagacatga gcattggaat gacagttatc aaaaacaaca tgataaataa tgatctcggt 1620cccgccacgg cacaaatggc actccaactc ttcattaagg attatcggta cacataccgg 1680tgtcaaagag gcgataccca gatacaaacc agaagatcct ttgagttgaa gaaactgtgg 1740gaacagactc gatcaaagac tggtctactg gtatcagatg ggggtccaaa cctatacaac 1800atcagaaacc tacacatccc ggaagtctgt ttgaaatggg agctgatgga tgaagattat 1860aaagggaggc tatgtaatcc attgaatcct ttcgttagcc acaaagaaat tgaatcagtg 1920aacagtgcag tagtaatgcc tgcgcatggc cctgccaaaa gcatggagta tgatgctgtt 1980gcaacaacac actcttggat ccccaagagg aaccggtcca tattgaacac aagtcaaagg 2040ggaatactcg aagatgagca gatgtatcag aaatgctgca acctgtttga aaaattcttc 2100cccagcagct catacagaar accagtcgga atttctagta tggttgaggc catggtgtcc 2160agggcccgca ttgatgcacg aattgacttc gaatctggac ggataaagaa ggatgagttc 2220gctgagatca tgaagatctg ttccaccatt gaagagctca gacggcaaaa a 2271

What is claimed:
 1. An isolated equine influenza nucleic acid moleculeselected from the group consisting of: a. an isolated nucleic acidmolecule that encodes a protein comprising amino acid sequence SEQ IDNO:48; and b. an isolated nucleic acid molecule fully complementary to anucleic acid molecule of (a); wherein said nucleic acid molecule of (a)or (b) is not an entire equine influenza virus genome.
 2. The nucleicacid molecule of claim 1, wherein said nucleic acid molecule comprisesnucleic acid sequence SEQ ID NO:47, and a nucleic acid moleculecomprising a nucleic acid sequence which is fully complementary to anyof said nucleic acid sequences.
 3. A nucleic acid molecule of claim 1,wherein said nucleic acid molecule encodes protein.
 4. A nucleic acidmolecule of claim 1, wherein said nucleic acid molecule encodes proteinPei_(ca1)PB2₇₅₉.